Detection of target analytes using particles and electrodes

ABSTRACT

The invention relates to the use of particles comprising binding ligands and electron transfer moieties (ETMs). Upon binding of a target analyte, a particle and a reporter composition are associated and transported to an electrode surface. The ETMs are then detected, allowing the presence or absence of the target analyte to be determined.

FIELD OF THE INVENTION

The invention relates to the use of particles comprising binding ligandsand electron transfer moieties (ETMs). Upon binding of a target analyte,a particle and a reporter composition are associated and transported toan electrode surface. The ETMs are then detected, allowing the presenceor absence of the target analyte to be determined.

BACKGROUND OF THE INVENTION

There are a number of assays and sensors for the detection of thepresence and/or concentration of specific substances in fluids andgases. Many of these rely on specific ligand/antiligand reactions as themechanism of detection. That is, pairs of substances (i.e. the bindingpairs or ligand/antiligands) are known to bind to each other, whilebinding little or not at all to other substances. This has been thefocus of a number of techniques that utilize these binding pairs for thedetection of the complexes. These generally are done by labeling onecomponent of the complex in some way, so as to make the entire complexdetectable, using, for example, radidisotopes, fluorescent and otheroptically active molecules, enzymes, etc.

Other assays rely on electronic signals for detection. Of particularinterest are biosensors. At least two types of biosensors are known;enzyme-based or metabolic biosensors and binding or bioaffinity sensors.See for example U.S. Pat. Nos. 4,713,347; 5,192,507; 4,920,047;3,873,267; and references disclosed therein. While some of these knownsensors use alternating current (AC) techniques, these techniques aregenerally limited to the detection of differences in bulk (ordielectric) impedance, and rely on the use of mediators in solution toshuttle the charge to the electrode.

Recently, there have been several preliminary reports on the use of veryshort connections between a binding ligand and the electrode, for directdetection, i.e. without the use of mediators. See Lötzbeyer et al.,Bioelectrochemistry and Bioenergetics 42:1-6 (1997); Dong et al.,Bioelectrochemistry and Bioenergetics 42:7-13 (1997).

In addition, there are a number of reports of self-assembled monolayersof conjugated oligomers on surfaces such as gold. See for example Cyganet al., J. Am. Chem. Soc. 120:2721 (1998).

In addition, Charych et al. report on the direct colormetric detectionof a receptor-ligand interaction using 3 bilayer assembly (Science261:585 (1993).

PCT applications WO 95/15971, PCT/US96/09769, PCT/US97/09739, WO96/40712and PCT/US97/20014 describe novel compositions comprising nucleic acidscontaining electron transfer moieties, including electrodes, which allowfor novel detection methods of nucleic acid hybridization.

In addition, there are a number of sensors that rely on the use ofparticles, including magnetic particles, particularly forelectrochemiluminescence detection. See U.S. Pat. Nos. 5,746,974;5,770,459; 5,779,976; 4,731,337; 4,115,535; 4,777,145; 4,945,045;4,978,610; 5,705,402; 4,910,148; 5,512,439; 5,585,241; and 5,609,907;and WO 90/14891; WO 90/05301; WO 92/14139; and WO 90/06044.

Finally, there are reports that bring particles together using nucleicacids for archetectural reasons. See Mirkin et al., Nature 382:607(1996); Mirkin et al., Science 277:1078 (1997); Storhoff et al., J. Am.Chem. Soc. 120:1959 (1998); and WO 98/10289.

SUMMARY OF THE INVENTION

In accordance with the objects outlined above, the present inventionprovides compositions comprising a gold colloid particle comprising atleast one ETM. The colloid can further comprise a self-assembledmonolayer (SAM). The compositions can further comprise an electrode,that may also contain a SAM.

In an additional aspect, the invention provides compositions comprisinggold colloid particles comprising a SAM, a capture probe, anamplification sequence; and a label probe hybridized to theamplification sequence, wherein the label probe comprises at least onecovalently attached ETM.

In an additional aspect, the invention provides compositions comprisinga transport composition comprising a first binding partner that directlyor indirectly binds a target analyte, and a reporter composition. Thereporter composition comprises a second binding partner that directly orindirectly binds the target analyte and a plurality of electron transfermoieties (ETMs). At least one of the transport and the reportercompositions is a particle. Upon introduction of the target analyte, thetransport composition and the reporter composition are associated. Thesecompositions may also comprise SAMs, and an electrode, optionallycontaining a SAM, can be included.

In a further aspect, the invention provides methods of detecting atarget analyte in a sample. The methods comprise adding the sample to acomposition as outlined above, such that the target analyte binds to thetransport composition and the reporter composition to form an assaycomplex. The assy complex is transported to the electrode, and thepresence or absence of the ETMs is detected.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1G depict a variety of different embodiments of the invention.Many of the figures depict nucleic acids as the binding ligands, targetmoieties, and extender moieties, but non-nucleic acid embodiments canalso be used in a similar manner. FIG. 1A shows an assay complexcomprising a transport particle 5 with a first binding ligand 10. Anoptional SAM of moieties 45 that can be either conductive oligomers orinsulators may also be present. The target analyte 20 binds to firstbinding ligand 10 and the second binding ligand 25, attached to thereporter particle 30. The reporter particle 30 comprises a plurality ofETMs 40, preferably linked to the reporter particle 30 via conductiveoligomers 35. A SAM of moieties 45 may also optionally be present. Thebinding ligands 10 and 25 can be attached via attachment linkers 60, notshown, that can be a conductive oligomer, an insulator, or othermoieties, as outlined herein. FIG. 1B is similar, but utilizes anextender moiety 50; as will be appreciated by those in the art, theextender moiety 50 may also be placed between the target 20 and thefirst binding ligand 25. In addition, the extender moiety (sometimesreferred to herein as an amplifier probe) may also contain multipleamplification sequences for attachment of reporter compositions; thus nis an integer of at least one. FIG. 1C depicts the use of a capturebinding ligand 55 attached to the transport particle 5 via an attachmentlinker 60. Additional embodiments for the attachment of nucleic acids tothe electrode surface are shown in FIG. 2. The capture binding ligand 55binds to a capture binding partner 65 attached to electrode 85 via anattachment linker 60. The electrode comprises conductive oligomers 70and optional insulators 80. FIG. 1D depicts the use of a non-particletransport composition comprising a capture binding ligand 55 attachedvia an optional linker 90 (that can be an attachment linker) to thecapture binding ligand 10. FIG. 1E depicts the use of the target analyteas the capture binding moiety; the target 20 binds to the capturebinding partner 65, attached to the electrode 85 via an attachmentlinker 60. The second binding ligand 25 binds to a portion of the target20. Depending on the length (i.e. when the target analyte is a nucleicacid) or size of the target analyte, a plurality n of reportercompositions can be used; thus n is an integer of at least one. FIG. 1Fdepicts the case wherein two reporter particles are used; what isimportant in this embodiment is that aggregation does not occur to anappreciable extent in the absence of the target 20, and that generallythe two second binding ligands 25 each recognize a different part of thetarget 20, such that a single target will bring together at least tworeporter compositions. FIG. 1G depicts the use of a non-particlereporter composition, using a recruitment linker 90, as outlined herein.FIG. 1H depicts the use of a reporter particle 30 that has both a secondbinding ligand 25 and a recruitment linker 90.

FIGS. 2A, 2B and 2C depict three preferred embodiments for attaching atarget sequence to the electrode. Although generally depicted as nucleicacids, non-nucleic acid embodiments are also useful. FIG. 2A depicts atarget sequence 120 hybridized to a capture probe 100 linked via aattachment linker 106, which as outlined herein may be either aconductive oligomer or an insulator. The electrode 105 comprises amonolayer of passivation agent 107, which can comprise conductiveoligomers (herein depicted as 108) and/or insulators (herein depicted as109). As for all the embodiments depicted in the figures, n is aninteger of at least 1, although as will be appreciated by those in theart, the system may not utilize a capture probe at all (i.e. n is zero),although this is generally not preferred. The upper limit of n willdepend on the length of the target sequence and the requiredsensitivity. FIG. 2B depicts the use of a single capture extender probe110 with a first portion 111 that will hybridize to a first portion ofthe target sequence 120 and a second portion that will hybridize to thecapture probe 100. FIG. 2C depicts the use of two capture extenderprobes 110 and 130. The first capture extender probe 110 has a firstportion 111 that will hybridize to a first portion of the targetsequence 120 and a second portion 112 that will hybridize to a firstportion 102 of the capture probe 100. The second capture extender probe130 has a first portion 132 that will hybridize to a second portion ofthe target sequence 120 and a second portion 131 that will hybridize toa second portion 101 of the capture probe 100.

FIGS. 3A, 3B, 3C, 3D and 3E depict different possible configurations oflabel probes and attachments of ETMs. The figures depict a secondbinding ligand that is a hybridizable nucleic acid, but as will beappreciated by those in the art, the second binding ligand can benon-nucleic acid as well. In FIGS. 3A-C, the recruitment linker isnucleic acid; in FIGS. 3D and E, is not. A=nucleoside replacement;B=attachment to a base; C=attachment to a ribose; D=attachment to aphosphate; E=metallocene polymer (although as described herein, this canbe a polymer of other ETMs as well), attached to a base, ribose orphosphate (or other backbone analogs); F=dendrimer structure, attachedvia a base, ribose or phosphate (or other backbone analogs);G=attachment via a “branching” structure, through base, ribose orphosphate (or other backbone analogs); H=attachment of metallocene (orother ETM) polymers; I=attachment via a dendrimer structure;J=attachment using standard linkers.

FIG. 4 depicts a schematic of the synthesis of simultaneousincorporation of multiple ETMs into a nucleic acid, using the N17“branch” point nucleoside.

FIG. 5 depicts a schematic of an alternate method of adding largenumbers of ETMs simultaneously to a nucleic acid using a “branch” pointphosphoramidite, in this case utilizing three branch points (althoughtwo branch points are also possible; see for example FIG. 1N) as isknown in the art. As will be appreciated by those in the art, each endpoint can contain any number of ETMs.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides novel analytical biosensors that can beused to sensitively detect target analytes. In one embodiment, thesystem provides two basic components: (1) a first component (generallybut not always a particle) that can bind a target analyte and that canbe used to transport an assay complex comprising the first component toan electrode, and (2) a reporter composition (which may or may not be aparticle as well) that can bind a target analyte and comprises electrontransfer moieties (ETMs). The two components are brought together by thedirect or indirect binding of a target analyte. That is, a single targetanalyte directly or indirectly binds both a first binding ligandattached to the first component and a second binding ligand attached tothe reporter composition; this forms an assay complex. The firstcomponent can be used to transport the assay complex to an electrode fordetection of the ETMs. This may be done in a variety of ways; forexample, when the first component is a particle, transport can occureither magnetically, when the particle is a magnetic particle, or viagravity or other techniques based on the specific gravity or density ofthe particle in relation to the solution. In some embodiments, bothcomponents are particles and the aggregation of the particles usingtarget analytes results in transport to the electrode. Alternatively,the first component may utilize a capture moiety for attachment to anelectrode that comprises a capture binding ligand. Once the assaycomplexes are formed, the presence or absence of the ETMs are detectedusing the electrode as is described below and in U.S. Pat. Nos.5,591,578; 5,824,473; 5,770,369; 5,705,348 and 5,780,234; U.S. Ser. Nos.08/911,589; 09/135,183; 09/306,653; 09/134,058; 09/295,691; 09/238,351;09/245,105, 60/145,912 and 09/338,726; and PCT applications WO98/20162;PCT US99/01705; PCT US99/01703; PCT US99/10104, all of which areexpressly incorporated herein by reference in their entirety.

In general, there are two basic detection mechanisms. In a preferredembodiment, detection of an ETM is based on electron transfer throughthe stacked n-orbitals of double stranded nucleic acid. This basicmechanism is described in U.S. Pat. Nos. 5,591,578, 5,770,369,5,705,348, 5,824,473 and 5,780,234 and WO98/20162, all of which areexpressly incorporated by reference, and is termed “mechanism-1” herein.Briefly, previous work has shown that electron transfer can proceedrapidly through the stacked n-orbitals of double stranded nucleic acid,and significantly more slowly through single-stranded nucleic acid.Accordingly, this can serve as the basis of an assay. Thus, by addingETMs (either covalently to one of the strands or non-covalently to thehybridization complex through the use of hybridization indicators,described below) to a nucleic acid that is attached to a detectionelectrode via a conductive oligomer, electron transfer between the ETMand the electrode, through the nucleic acid and conductive oligomer, maybe detected.

Alternatively, ETMs can be directly detected on a surface of amonolayer. That is, the electrons from the ETMs need not travel throughthe stacked n orbitals in order to generate a signal. As above, in thisembodiment, the detection electrode preferably comprises aself-assembled monolayer (SAM) that serves to shield the electrode fromredox-active species in the sample. In this embodiment, the presence ofETMs on the surface of a SAM, that has been formulated to compriseslight “defects” (sometimes referred to herein as “microconduits”,“nanoconduits” or “electroconduits”) can be directly detected. Thisbasic idea is termed “mechanism-2” herein. Essentially, theelectroconduits allow particular ETMs access to the surface. Withoutbeing bound by theory, it should be noted that the configuration of theelectroconduit depends in part on the ETM chosen. For example, the useof relatively hydrophobic ETMs allows the use of hydrophobicelectroconduit forming species, which effectively exclude hydrophilic orcharged ETMs. Similarly, the use of more hydrophilic or charged speciesin the SAM may serve to exclude hydrophobic ETMs.

It should be noted that these defects are to be distinguished from“holes” that allow direct contact of sample components with thedetection electrode. As is more fully outlined below, theelectroconduits can be generated in several general ways, including butnot limited to the use of rough electrode surfaces, such as goldelectrodes formulated on PC circuit boards; or the inclusion of at leasttwo different species in the monolayer, i.e. using a “mixed monolayer”,at least one of which is a electroconduit-forming species (EFS). Thut,upon binding of a target analyte, a binding ligand comprising an ETM isbrought to the surface, and detection of the ETM can proceed, putativelythrough the “electroconduits” to the electrode. Essentially, the role ofthe SAM comprising the defects is to allow contact of the ETM with theelectronic surface of the electrode, while still providing the benefitsof shielding the electrode from solution components and reducing theamount of non-specific binding to the electrodes. Viewed differently,the role of the binding ligand is to provide specificity for arecruitment of ETMs to the surface, where they can be directly detected.

Thus, in either embodiment, an assay complex is formed that contains anETM, which is then detected using the detection electrode. The inventionthus provides assay complexes that minimally comprise a target analyte.“Assay complex” herein is meant the collection of binding complexes,e.g. nucleic acids, including probes and targets, that contains at leastone ETM and thus allows detection. The composition of the assay complexdepends on the use of the different probe components outlined herein.

Accordingly, the present invention provides methods and compositionsuseful in the detection of target analytes in samples. As will beappreciated by those in the art, the sample solution may comprise anynumber of things, including, but not limited to, bodily fluids(including, but not limited to, blood, urine, serum, lymph, saliva, analand vaginal secretions, perspiration and semen, of virtually anyorganism, with mammalian samples being preferred and human samples beingparticularly preferred); environmental samples (including, but notlimited to, air, agricultural, water and soil samples); biologicalwarfare agent samples; research samples (i.e. in the case of nucleicacids, the sample may be the products of an amplification reaction,including both target and signal amplification as is generally describedin PCT/US99/01705, such as PCR amplification reaction); purifiedsamples, such as purified genomic DNA, RNA, proteins, etc.; raw samples(bacteria, virus, genomic DNA, etc.); as will be appreciated by those inthe art, virtually any experimental manipulation may have been done onthe sample.

The methods are directed to the detection of target analytes. By “targetanalyte” or “analyte” or grammatical equivalents herein is meant anymolecule or compound to be detected and that can bind to a bindingspecies, defined below. Suitable analytes include, but not limited to,small chemical molecules such as environmental or clinical chemical orpollutant or biomolecule, including, but not limited to, pesticides,insecticides, toxins, therapeutic and abused drugs, hormones,antibiotics, antibodies, organic materials, etc. Suitable biomoleculesinclude, but are not limited to, proteins (including enzymes,immunoglobulins and glycoproteins), nucleic acids, lipids, lectins,carbohydrates, hormones, whole cells (including procaryotic (such aspathogenic bacteria) and eucaryotic cells, including mammalian tumorcells), viruses, spores, etc. Particularly preferred analytes areproteins including enzymes; drugs, cells; antibodies; antigens; cellularmembrane antigens and receptors (neural, hormonal, nutrient, and cellsurface receptors) or their ligands.

By “proteins” or grammatical equivalents herein is meant proteins,oligopeptides and peptides, and analogs, including proteins containingnon-naturally occurring amino acids and amino acid analogs, andpeptidomimetic structures.

As will be appreciated by those in the art, a large number of analytesmay be detected using the present methods; basically, any target analytefor which a binding ligand, described below, may be made may be detectedusing the methods of the invention.

In a preferred embodiment, the target analytes are nucleic acids. By“nucleic acid” or “oligonucleotide” or grammatical equivalents hereinmeans at least two nucleotides covalently linked together. A nucleicacid of the present invention will generally contain phosphodiesterbonds, although in some cases, as outlined below, nucleic acid analogsare included that may have alternate backbones, comprising, for example,phosphoramide (Beaucage et al., Tetrahedron 49(10):1925 (1993) andreferences therein; Letsinger, J. Org. Chem. 35:3800 (1970); Sprinzl etal., Eur. J. Biochem. 81:579 (1977); Letsinger et al., Nucl. Acids Res.14:3487 (1986); Sawai et al, Chem. Lett. 805 (1984), Letsinger et al.,J. Am. Chem. Soc. 110:4470 (1988); and Pauwels et al., Chemica Scripta26:141 91986)), phosphorothioate (Mag et al., Nucleic Acids Res. 19:1437(1991); and U.S. Pat. No. 5,644,048), phosphorodithioate (Briu et al.,J. Am. Chem. Soc. 111:2321 (1989), O-methylphosphoroamidite linkages(see Eckstein, Oligonucleotides and Analogues: A Practical Approach,Oxford University Press), and peptide nucleic acid backbones andlinkages (see Egholm, J. Am. Chem. Soc. 114:1895 (1992); Meier et al.,Chem. Int. Ed. Engl. 31:1008 (1992); Nielsen, Nature, 365:566 (1993);Carlsson et al., Nature 380:207 (1996), all of which are incorporated byreference). Other analog nucleic acids include those with positivebackbones (Denpcy et al., Proc. Natl. Acad. Sci. USA 92:6097 (1995);non-ionic backbones (U.S. Pat. Nos. 5,386,023, 5,637,684, 5,602,240,5,216,141 and 4,469,863; Kiedrowshi et al., Angew. Chem. Intl. Ed.English 30:423 (1991); Letsinger et al., J. Am. Chem. Soc. 110:4470(1988); Letsinger et al., Nucleoside & Nucleotide 13:1597 (1994);Chapters 2 and 3, ASC Symposium Series 580, “Carbohydrate Modificationsin Antisense Research”, Ed. Y. S. Sanghui and P. Dan Cook; Mesmaeker etal., Bioorganic & Medicinal Chem. Lett. 4:395 (1994); Jeffs et al., J.Biomolecular NMR 34:17 (1994); Tetrahedron Lett. 37:743 (1996)) andnon-ribose backbones, including those described in U.S. Pat. Nos.5,235,033 and 5,034,506, and Chapters 6 and 7, ASC Symposium Series 580,“Carbohydrate Modifications in Antisense Research”, Ed. Y. S. Sanghuiand P. Dan Cook. Nucleic acids containing one or more carbocyclic sugarsare also included within the definition of nucleic acids (see Jenkins etal., Chem. Soc. Rev. (1995) pp 169-176). Several nucleic acid analogsare described in Rawls, C & E News Jun. 2, 1997 page 35. All of thesereferences are hereby expressly incorporated by reference. Thesemodifications of the ribose-phosphate backbone may be done to facilitatethe addition of ETMs, or to increase the stability and half-life of suchmolecules in physiological environments.

As will be appreciated by those in the art, all of these nucleic acidanalogs may find use in the present invention. In addition, mixtures ofnaturally occurring nucleic acids and analogs can be made; for example,at the site of conductive oligomer or ETM attachment, an analogstructure may be used. Alternatively, mixtures of different nucleic acidanalogs, and mixtures of naturally occurring nucleic acids and analogsmay be made.

Particularly preferred are peptide nucleic acids (PNA) which includespeptide nucleic acid analogs. These backbones are substantiallynon-ionic under neutral conditions, in contrast to the highly chargedphosphodiester backbone of naturally occurring nucleic acids. Thisresults in two advantages. First, the PNA backbone exhibits improvedhybridization kinetics. PNAs have larger changes in the meltingtemperature (Tm) for mismatched versus perfectly matched basepairs. DNAand RNA typically exhibit a 2-4° C. drop in Tm for an internal mismatch.With the non-ionic PNA backbone, the drop is closer to 7-9° C.Similarly, due to their non-ionic nature, hybridization of the basesattached to these backbones is relatively insensitive to saltconcentration. This is particularly advantageous in the systems of thepresent invention, as a reduced salt hybridization solution has a lowerFaradaic current than a physiological salt solution (in the range of 150mM).

The nucleic acids may be single stranded or double stranded, asspecified, or contain portions of both double stranded or singlestranded sequence. The nucleic acid may be DNA, both genomic and cDNA,RNA or a hybrid, where the nucleic acid contains any combination ofdeoxyribo- and ribo-nucleotides, and any combination of bases, includinguracil, adenine, thymine, cytosine, guanine, inosine, xathaninehypoxathanine, isocytosine, isoguanine, etc. A preferred embodimentutilizes isocytosine and isoguanine in nucleic acids designed to becomplementary to other probes, rather than target sequences, as thisreduces non-specific hybridization, as is generally described in U.S.Pat. No. 5,681,702. As used herein, the term “nucleoside” includesnucleotides as well as nucleoside and nucleotide analogs, and modifiednucleosides such as amino modified nucleosides. In addition,“nucleoside” includes non-naturally occurring analog structures. Thusfor example the individual units of a peptide nucleic acid, eachcontaining a base, are referred to herein as a nucleoside.

Thus, in a preferred embodiment, the target analyte is a targetsequence. The term “target sequence” or “target nucleic acid” orgrammatical equivalents herein means a nucleic acid sequence on a singlestrand of nucleic acid. The target sequence may be a portion of a gene,a regulatory sequence, genomic DNA, cDNA, RNA including mRNA and rRNA,or others. As is outlined herein, the target sequence may be a targetsequence from a sample, or a secondary target such as a product of anamplification reaction, etc. It may be any length, with theunderstanding that longer sequences are more specific. As will beappreciated by those in the art, the complementary target sequence maytake many forms. For example, it may be contained within a largernucleic acid sequence, i.e. all or part of a gene or mRNA, a restrictionfragment of a plasmid or genomic DNA, among others. As is outlined morefully below, probes are made to hybridize to target sequences todetermine the presence or absence of the target sequence in a sample.Generally speaking, this term will be understood by those skilled in theart. The target sequence may also be comprised of different targetdomains; for example, a first target domain of the sample targetsequence may hybridize to a capture probe or a portion of captureextender probe, a second target domain may hybridize to a portion of anamplifier probe, a label probe, or a different capture or captureextender probe, etc. The target domains may be adjacent or separated asindicated. Unless specified, the terms “first” and “second” are notmeant to confer an orientation of the sequences with respect to the5′-3′ orientation of the target sequence. For example, assuming a 5′-3′orientation of the complementary target sequence, the first targetdomain may be located, either 5′ to the second domain, or 3′ to thesecond domain.

In a preferred embodiment, the methods of the invention are used todetect pathogens such as bacteria. In this embodiment, preferred targetsequences include rRNA, as is generally described in U.S. Pat. Nos.4,851,330; 5,288,611; 5,723,597; 6,641,632; 5,738,987; 5,830,654;5,763,163; 5,738,989; 5,738,988; 5,723,597; 5,714,324; 5,582,975;5,747,252; 5,567,587; 5,558,990; 5,622,827; 5,514,551; 5,501,951;5,656,427; 5,352,579; 5,683,870; 5,374,718; 5,292,874; 5,780,219;5,030,557; and 5,541,308, all of which are expressly incorporated byreference.

As will be appreciated by those in the art, a large number of analytesmay be detected using the present methods; basically, any target analytefor which a binding ligand, described below, may be made may be detectedusing the methods of the invention. While many of the techniquesdescribed below exemplify nucleic acids as the target analyte, those ofskill in the art will recognize that other target analytes can bedetected using the same systems.

If required, the target analyte is prepared using known techniques. Forexample, the sample may be treated to lyse the cells, using known lysisbuffers, electroporation, etc., with purification and/or amplificationas needed, as will be appreciated by those in the art. When the targetanalyte is a nucleic acid, the target sequence may be amplified asrequired; suitable amplification techniques are outlined in PCTUS99/01705, hereby expressly incorporated by reference. In addition,techniques to increase the amount or rate of hybridization can also beused; see for example U.S. Ser. No. 09/338,726; filed Jun. 23, 1999,hereby incorporated by reference.

All of these techniques rely on the formation of assay complexes on asurface, frequently an electrode, as is described herein. The assaycomplex preferably includes at least one particle, generally a goldcolloid particle, that is used either as a transport particle or areporter particle, or both. The assay complex further comprises at leastone electron transfer moiety (ETM), that is also either directly orindirectly attached to the assay complex. Once the assay complexes areformed, the presence or absence of the ETMs are detected as is describedbelow and in U.S. Pat. Nos. 5,591,578; 5,824,473; 5,770,369; 5,705,348and 5,780,234; U.S. Ser. Nos. 08/911,589; 09/135,183; 09/306,653;09/134,058; 09/295,691; 09/238,351; 60/145,912 and 09/245,105: and PCTapplications WO98/20162; PCT US99/01705; PCT US99/01703; PCT US99/14191;PCT US99/10104, all of which are expressly incorporated herein byreference in their entirety.

Accordingly, the present invention provides methods and compositionsuseful in the detection of target analytes, including nucleic acids. Aswill be appreciated by those in the art, the compositions of theinvention can take on a wide variety of configurations, as is generallyoutlined in the Figures and described below. It should be noted thatwhile the discussion below focuses on the detection of nucleic acids,other target analytes can be used in any of the systems describedherein.

Thus, in a preferred embodiment, the compositions comprise an electrode.By “electrode” herein is meant a composition, which, when connected toan electronic device, is able to sense a current or charge and convertit to a signal. Alternatively an electrode can be defined as acomposition which can apply a potential to and/or pass electrons to orfrom species in the solution. Thus, an electrode is an ETM as describedherein. Preferred electodes are known in the art and include, but arenot limited to, certain metals and their oxides, including gold;platinum; palladium; silicon; aluminum; metal oxide electrodes includingplatinum oxide, titanium oxide, tin oxide, indium tin oxide, palladiumoxide, silicon oxide, aluminum oxide, molybdenum oxide (Mo₂O₆), tungstenoxide (WO₃) and ruthenium oxides; and carbon (including glassy carbonelectrodes, graphite and carbon paste). Preferred electrodes includegold, silicon, carbon and metal oxide electrodes, with gold beingparticularly preferred.

The electrodes described herein are depicted as a flat surface, which isonly one of the possible conformations of the electrode and is forschematic purposes only. The conformation of the electrode will varywith the detection method used. For example, flat planar electrodes maybe preferred for optical detection methods, or when arrays of nucleicacids are made, thus requiring addressable locations for both synthesisand detection. Alternatively, for single probe analysis, the electrodemay be in the form of a tube, with the SAMs comprising conductiveoligomers and nucleic acids bound to the inner surface. This allows amaximum of surface area containing the nucleic acids to be exposed to asmall volume of sample.

In addition, the geometry of the system may alter with the transportmechanism used. For example, when aggregation of the particles is usedto transport the ETMs to the electrode surface, the electrode surfaceneeds to be at the bottom. However, other systems utlizing differenttransport mechanisms can have the electrode surface be anywhere.

In a preferred embodiment, the detection electrodes are formed on asubstrate. In addition, the discussion herein is generally directed tothe formation of gold electrodes, but as will be appreciated by those inthe art, other electrodes can be used as well. The substrate cancomprise a wide variety of materials, as will be appreciated by those inthe art, with printed circuit board (PCB) materials being particularlypreferred. Thus, in general, the suitable substrates include, but arenot limited to, fiberglass, teflon, ceramics, glass, silicon, mica,plastic (including acrylics, polystyrene and copolymers of styrene andother materials, polypropylene, polyethylene, polybutylene,polycarbonate, polyurethanes, Teflon™, and derivatives thereof, etc.),GETEK (a blend of polypropylene oxide and fiberglass), etc.

In general, preferred materials include printed circuit board materials.Circuit board materials are those that comprise an insulating substratethat is coated with a conducting layer and processed using lithographytechniques, particularly photolithography techniques, to form thepatterns of electrodes and interconnects (sometimes referred to in theart as interconnections or leads). The insulating substrate isgenerally, but not always, a polymer. As is known in the art, one or aplurality of layers may be used, to make either “two dimensional” (e.g.all electrodes and interconnections in a plane) or “three dimensional”(wherein the electrodes are on one surface and the interconnects may gothrough the board to the other side) boards. Three dimensional systemsfrequently rely on the use of drilling or etching, followed byelectroplating with a metal such as copper, such that the “throughboard” interconnections are made. Circuit board materials are oftenprovided with a foil already attached to the substrate, such as a copperfoil, with additional copper added as needed (for example forinterconnections), for example by electroplating. The copper surface maythen need to be roughened, for example through etching, to allowattachment of the adhesion layer.

Accordingly, in a preferred embodiment, the present invention providesbiochips (sometimes referred to herein “chips”) that comprise substratescomprising a plurality of electrodes, preferably gold electrodes. Thenumber of electrodes is as outlined for arrays. Each electrodepreferably comprises a self-assembled monolayer as outlined herein. In apreferred embodiment, one of the monolayer-forming species comprises acapture ligand as outlined herein. In addition, each electrode has aninterconnection, that is attached to the electrode at one end and isultimately attached to a device that can control the electrode. That is,each electrode is independently addressable.

The substrates can be part of a larger device comprising a detectionchamber that exposes a given volume of sample to the detectionelectrode. Generally, the detection chamber ranges from about 1 mL to 1ml, with about 10 μL to 500 μL being preferred. As will be appreciatedby those in the art, depending on the experimental conditions and assay,smaller or larger volumes may be used.

In some embodiments, the detection chamber and electrode are part of acartridge that can be placed into a device comprising electroniccomponents (an AC/DC voltage source, an ammeter, a processor, a read-outdisplay, temperature controller, light source, etc.). In thisembodiment, the interconnections from each electrode are positioned suchthat upon insertion of the cartridge into the device, connectionsbetween the electrodes and the electronic components are established.

Detection electrodes on circuit board material (or other substrates) aregenerally prepared in a wide variety of ways. In general, high puritygold is used, and it may be deposited on a surface via vacuum depositionprocesses (sputtering and evaporation) or solution deposition(electroplating or electroless processes). When electroplating is done,the substrate must initially comprise a conductive material; fiberglasscircuit boards are frequently provided with copper foil. Frequently,depending on the substrate, an adhesion layer between the substrate andthe gold in order to insure good mechanical stability is used. Thus,preferred embodiments utilize a deposition layer of an adhesion metalsuch as chromium, titanium, titanium/tungsten, tantalum, nickel orpalladium, which can be deposited as above for the gold. Whenelectroplated metal (either the adhesion metal or the electrode metal)is used, grain refining additives, frequently referred to in the tradeas brighteners, can optionally be added to alter surface depositionproperties. Preferred brighteners are mixtures of organic and inorganicspecies, with cobalt and nickel being preferred.

In general, the adhesion layer is from about 100 Å thick to about 25microns (1000 microinches). The If the adhesion metal iselectrochemically active, the electrode metal must be coated at athickness that prevents “bleed-through”; if the adhesion metal is notelectrochemically active, the electrode metal may be thinner. Generally,the electrode metal (preferably gold) is deposited at thicknessesranging from about 500 Å to about 5 microns (200 microinches), with fromabout 30 microinches to about 50 microinches being preferred. Ingeneral, the gold is deposited to make electrodes ranging in size fromabout 5 microns to about 5 mm in diameter, with about 100 to 250 micronsbeing preferred. The detection electrodes thus formed are thenpreferably cleaned and SAMs added, as is discussed below.

Thus, the present invention provides methods of making a substratecomprising a plurality of gold electrodes. The methods first comprisecoating an adhesion metal, such as nickel or palladium (optionally withbrightener), onto the substrate. Electroplating is preferred. Theelectrode metal, preferably gold, is then coated (again, withelectroplating preferred) onto the adhesion metal. Then the patterns ofthe device, comprising the electrodes and their associatedinterconnections are made using lithographic techniques, particularlyphotolithographic techniques as are known in the art, and wet chemicaletching. Frequently, a non-conductive chemically resistive insulatingmaterial such as solder mask or plastic is laid down using thesephotolithographic techniques, leaving only the electrodes and aconnection point to the leads exposed; the leads themselves aregenerally coated.

The methods continue with the addition of SAMs as are described below.In a preferred embodiment, drop deposition techniques are used to addthe required chemistry, i.e. the monolayer forming species, one of whichis preferably a capture ligand comprising species. Drop depositiontechniques are well known for making “spot” arrays. This is done to adda different composition to each electrode, i.e. to make an arraycomprising different capture ligands. Alternatively, the SAM species maybe identical for each electrode, and this may be accomplished using adrop deposition technique or the immersion of the entire substrate or asurface of the substrate into the solution.

Thus, in a preferred embodiment, the electrode comprises a monolayer,comprising electroconduit forming species (EFS). As outlined herein, theefficiency of target analyte binding (for example, oligonucleotidehybridization) may increase when the analyte is at a distance from theelectrode. Similarly, non-specific binding of biomolecules, includingthe target analytes, to an electrode is generally reduced when amonolayer is present. Thus, a monolayer facilitates the maintenance ofthe analyte away from the electrode surface. In addition, a monolayerserves to keep charged species away from the surface of the electrode.Thus, this layer helps to prevent electrical contact between theelectrodes and the ETMs, or between the electrode and charged specieswithin the solvent. Such contact can result in a direct “short circuit”or an indirect short circuit via charged species which may be present inthe sample. Accordingly, the monolayer is preferably tightly packed in auniform layer on the electrode surface, such that a minimum of “holes”exist. The monolayer thus serves as a physical barrier to block solventaccesibility to the electrode:

By “monolayer” or “self-assembled monolayer” or “SAM” herein is meant arelatively ordered assembly of molecules spontaneously chemisorbed on asurface, in which the molecules are oriented approximately parallel toeach other and roughly perpendicular to the surface. Each of themolecules includes a functional group that adheres to the surface, and aportion that interacts with neighboring molecules in the monolayer toform the relatively ordered array. A “mixed” monolayer comprises aheterogeneous monolayer, that is, where at least two different moleculesmake up the monolayer.

In general, the SAMs of the invention can be generated in a number ofways and comprise a number of different components, depending on theelectrode surface and the system used. For “mechanism-1” embodiments,preferred embodiments utilize two monolayer forming species: a monolayerforming species (including insulators or conductive oligomers) and aconductive oligomer species comprising the capture binding ligand,although as will be appreciated by those in the art, additionalmonolayer forming species can be included as well. For “mechanism-2”systems, the composition of the SAM depends on the detection electrodesurface. In general, two basic “mechanism-2” systems are described;detection electrodes comprising “smooth” surfaces, such as gold ballelectrodes, and those comprising “rough” surfaces, such as those thatare made using commercial processes on PC circuit boards. In general,without being bound by theory, it appears that monolayers made onimperfect surfaces, i.e. “rough” surfaces, spontaneously form monolayerscontaining enough electroconduits even in the absence of EFS, probablydue to the fact that the formation of a uniform monolayer on a roughsurface is difficult. “Smoother” surfaces, however, may require theinclusion of sufficient numbers of EFS to generate the electroconduits,as the uniform surfaces allow a more uniform monolayer to form. Again,without being bound by theory, the inclusion of species that disturb theuniformity of the monolayer, for example by including a rigid moleculein a background of more flexible ones, causes electroconduits. Thus“smooth” surfaces comprise monolayers comprising three components: aninsulator species, a EFS, and a species comprising the capture ligand,although in some circumstances, for example when the capture ligandspecies is included at high density, the capture ligand species canserve as the EFS. “Smoothness” in this context is not measuredphysically but rather as a function of an increase in the measuredsignal when EFS are included. That is, the signal from a detectionelectrode coated with monolayer forming species is compared to a signalfrom a detection electrode coated with monolayer forming speciesincluding a EFS. An increase indicates that the surface is relativelysmooth, since the inclusion of a EFS served to facilitate the access ofthe ETM to the electrode. It should also be noted that while thediscussion herein is mainly directed to gold electrodes andthiol-containing monolayer forming species, other types of electrodesand monolayer-forming species can be used.

It should be noted that the “electroconduits” of mechanism-2 systems donot result in direct contact of sample components with the electrodesurface; that is, the electroconduits are not large pores or holes thatallow physical access to the electrode. Rather, without being bound bytheory, it appears that the electroconduits allow certain types of ETMs,particularly hydrophobic ETMs, to penetrate sufficiently into themonolayer to allow detection. However, other types of redox activespecies, including some hydrophilic species, do not penentrate into themonolayer, even with electroconduits present. Thus, in general, redoxactive species that may be present in the sample do not give substantialsignals as a result of the electroconduits. While the exact system willvary with the composition of the SAM and the choice of the ETM, ingeneral, the test for a suitable SAM to reduce non-specific binding thatalso has sufficient electroconduits for E™ detection is to add eitherferrocene or ferrocyanide to the SAM; the former should give a signaland the latter should not.

Accordingly, in mechanism-1 systems, the monolayer comprises a firstspecies comprising a conductive oligomer comprising the capture bindingligand, as is more fully outlined below, and a second species comprisinga monolayer forming species, including either or both insulators orconductive oligomers.

In a preferred embodiment, the monolayer compriseselectroconduit-forming species. By “electroconduit-forming species” or“EFS” herein is meant a molecule that is capable of generatingsufficient electroconduits in a monolayer, generally of insulators suchas alkyl groups, to allow detection of ETMs at the surface. In general,EFS have one or more of the following qualities: they may be relativelyrigid molecules, for example as compared to an alkyl chain; they mayattach to the electrode surface with a geometry different from the othermonolayer forming species (for example, alkyl chains attached to goldsurfaces with thiol groups are thought to attach at roughly 45° angles,and phenyl-acetylene chains attached to gold via thiols are thought togo down at 90° angles); they may have a structure that stericallyinterferes or interrupts the formation of a tightly packed monolayer,for example through the inclusion of branching groups such as alkylgroups, or the inclusion of highly flexible species, such aspolyethylene glycol units; or they may be capable of being activated toform electroconduits; for example, photoactivatible species that can beselectively removed from the surface upon photoactivation, leavingelectroconduits.

Preferred EFS include conductive oligomers, as defined below, andphenyl-acetylene-polyethylene glycol species. However, in someembodiments, the EFS is not a conductive oligomer.

In a preferred embodiment, the monolayer comprises conductive oligomers.By “conductive oligomer” herein is meant a substantially conductingoligomer, preferably linear, some embodiments of which are referred toin the literature as “molecular wires”. By “substantially conducting”herein is meant that the oligomer is capable of transfering electrons at100 Hz. Generally, the conductive oligomer has substantially overlappingn-orbitals, i.e. conjugated n-orbitals, as between the monomeric unitsof the conductive oligomer, although the conductive oligomer may alsocontain one or more sigma (σ) bonds. Additionally, a conductive oligomermay be defined functionally by its ability to inject or receiveelectrons into or from an associated ETM. Furthermore, the conductiveoligomer is more conductive than the insulators as defined herein.Additionally, the conductive oligomers of the invention are to bedistinguished from electroactive polymers, that themselves may donate oraccept electrons.

In a preferred embodiment, the conductive oligomers have a conductivity,S, of from between about 10⁻⁶ to about 10⁴ Ω⁻¹˜cm⁻¹, with from about10⁻⁵ to about 10³ Ω⁻¹·cm⁻¹ being preferred, with these S values beingcalculated for molecules ranging from about 20 Å to about 200 Å. Asdescribed below, insulators have a conductivity S of about 10⁻⁷ Ω⁻¹·cm⁻¹or lower, with less than about 10⁻⁸ Ω⁻¹·cm⁻¹ being preferred. Seegenerally Gardner et al., Sensors and Actuators A 51 (1995) 57-66,incorporated herein by reference.

Desired characteristics of a conductive oligomer include highconductivity, sufficient solubility in organic solvents and/or water forsynthesis and use of the compositions of the invention, and preferablychemical resistance to reactions that occur i) during synthesis of thecomponents of the system, ii) during the attachment of the conductiveoligomer to an electrode, or iii) during detection assays. In addition,conductive oligomers that will promote the formation of self-assembledmonolayers are preferred.

The oligomers of the invention comprise at least two monomeric subunits,as described herein. As is described more fully below, oligomers includehomo- and hetero-oligomers, and include polymers.

In a preferred embodiment, the conductive oligomer has the structuredepicted in Structure 1:

As will be understood by those in the art, all of the structuresdepicted herein may have additional atoms or structures; i.e. theconductive oligomer of Structure 1 may be attached to ETMs, such aselectrodes, transition metal complexes, organic ETMs, and metallocenes,and to capture binding ligands, or to several of these. Unless otherwisenoted, the conductive oligomers depicted herein will be attached at theleft side to an electrode; that is, as depicted in Structure 1, the left“Y” is connected to the electrode as described herein. If the conductiveoligomer is to be attached to a nucleic acid, the right “Y”, if present,is attached to the nucleic acid, either directly or through the use of alinker, as is described herein.

In this embodiment, Y is an aromatic group, n is an integer from 1 to50, g is either 1 or zero, e is an integer from zero to 10, and m iszero or 1. When g is 1, B-D is a bond able to conjugate with neighboringbonds (herein referred to as a “conjugated bond”), preferably selectedfrom acetylene, B-D is a conjugated bond, preferably selected fromacetylene, alkene, substituted alkene, amide, azo, —C═N— (including—N═C—, —CR═N— and —N═CR—), —Si═Si—, and —Si═C— (including —C═Si—,—Si═CR— and —CR═Si—). When g is zero, e is preferably 1, D is preferablycarbonyl, or a heteroatom moiety, wherein the heteroatom is selectedfrom oxygen, sulfur, nitrogen, silicon or phosphorus. Thus, suitableheteroatom moieties include, but are not limited to, —NH and —NR,wherein R is as defined herein; substituted sulfur; sulfonyl (—SO₂—)sulfoxide (—SO—); phosphine oxide (—PO— and —RPO—); and thiophosphine(—PS— and —RPS—). However, when the conductive oligomer is to beattached to a gold electrode, as outlined below, sulfur derivatives arenot preferred.

By “aromatic group” or grammatical equivalents herein is meant anaromatic monocyclic or polycyclic hydrocarbon moiety generallycontaining 5 to 14 carbon atoms (although larger polycyclic ringsstructures may be made) and any carbocylic ketone or thioketonederivative thereof, wherein the carbon atom with the free valence is amember of an aromatic ring. Aromatic groups include arylene groups andaromatic groups with more than two atoms removed. For the purposes ofthis application aromatic includes heterocycle. “Heterocycle” or“heteroaryl” means an aromatic group wherein 1 to 5 of the indicatedcarbon atoms are replaced by a heteroatom chosen from nitrogen, oxygen,sulfur, phosphorus, boron and silicon wherein the atom with the freevalence is a member of an aromatic ring, and any heterocyclic ketone andthioketone derivative thereof. Thus, heterocycle includes thienyl,furyl, pyrrolyl, pyrimidinyl, oxalyl, indolyl, purinyl, quinolyl,isoquinolyl, thiazolyl, imidozyl, etc.

Importantly, the Y aromatic groups of the conductive oligomer may bedifferent, i.e. the conductive oligomer may be a heterooligomer. Thatis, a conductive oligomer may comprise a oligomer of a single type of Ygroups, or of multiple types of Y groups.

The aromatic group may be substituted with a substitution group,generally depicted herein as R. R groups may be added as necessary toaffect the packing of the conductive oligomers, i.e. R groups may beused to alter the association of the oligomers in the monolayer. Rgroups may also be added to 1) alter the solubility of the oligomer orof compositions containing the oligomers; 2) alter the conjugation orelectrochemical potential of the system; and 3) alter the charge orcharacteristics at the surface of the monolayer.

In a preferred embodiment, when the conductive oligomer is greater thanthree subunits, R groups are preferred to increase solubility whensolution synthesis is done. However, the R groups, and their positions,are chosen to minimally effect the packing of the conductive oligomerson a surface, particularly within a monolayer, as described below. Ingeneral, only small R groups are used within the monolayer, with largerR groups generally above the surface of the monolayer. Thus for examplethe attachment of methyl groups to the portion of the conductiveoligomer within the monolayer to increase solubility is preferred, withattachment of longer alkoxy groups, for example, C3 to C10, ispreferably done above the monolayer surface. In general, for the systemsdescribed herein, this generally means that attachment of stericallysignificant R groups is not done on any of the first two or threeoligomer subunits, depending on the average length of the moleculesmaking up the monolayer.

Suitable R groups include, but are not limited to, hydrogen, alkyl,alcohol, aromatic, amino, amido, nitro, ethers, esters, aldehydes,sulfonyl, silicon moieties, halogens, sulfur containing moieties,phosphorus containing moieties, and ethylene glycols. In the structuresdepicted herein, R is hydrogen when the position is unsubstituted. Itshould be noted that some positions may allow two substitution groups, Rand R′, in which case the R and R′ groups may be either the same ordifferent.

By “alkyl group” or grammatical equivalents herein is meant a straightor branched chain alkyl group, with straight chain alkyl groups beingpreferred. If branched, it may be branched at one or more positions, andunless specified, at any position. The alkyl group may range from about1 to about 30 carbon atoms (C1-C30), with a preferred embodimentutilizing from about 1 to about 20 carbon atoms (C1-C20), with about C1through about C12 to about C15 being preferred, and C1 to C5 beingparticularly preferred, although in some embodiments the alkyl group maybe much larger. Also included within the definition of an alkyl groupare cycloalkyl groups such as C5 and C6 rings, and heterocyclic ringswith nitrogen, oxygen, sulfur or phosphorus. Alkyl also includesheteroalkyl, with heteroatoms of sulfur, oxygen, nitrogen, and siliconebeing preferred. Alkyl includes substituted alkyl groups. By“substituted alkyl group” herein is meant an alkyl group furthercomprising one or more substitution moieties “R”, as defined above.

By “amino groups” or grammatical equivalents herein is meant —NH₂, —NHRand —NR₂ groups, with R being as defined herein.

By “nitro group” herein is meant an —NO₂ group.

By “sulfur containing moieties” herein is meant compounds containingsulfur atoms, including but not limited to, thia-, thio- andsulfo-compounds, thiols (—SH and —SR), and sulfides (—RSR—). By“phosphorus containing moieties” herein is meant compounds containingphosphorus, including, but not limited to, phosphines and phosphates. By“silicon containing moieties” herein is meant compounds containingsilicon.

By “ether” herein is meant an —O—R group. Preferred ethers includealkoxy groups, with —O—(CH₂)₂CH₃ and —O—(CH₂)₄CH₃ being preferred.

By “ester” herein is meant a —COOR group.

By “halogen” herein is meant bromine, iodine, chlorine, or fluorine.Preferred substituted alkyls are partially or fully halogenated alkylssuch as CF₃, etc.

By “aldehyde” herein is meant —RCHO groups.

By “alcohol” herein is meant —OH groups, and alkyl alcohols —ROH.

By “amido” herein is meant —RCONH— or RCONR— groups.

By “ethylene glycol” or “(poly)ethylene glycol” herein is meant a—(O—CH₂—CH₂)_(n)— group, although each carbon atom of the ethylene groupmay also be singly or doubly substituted, i.e. —(O—CR₂—CR₂)_(n)—, with Ras described above. Ethylene glycol derivatives with other heteroatomsin place of oxygen (i.e. —(N—CH₂—CH₂)— or —(S—CH₂—CH₂)_(n)—, or withsubstitution groups) are also preferred.

Preferred substitution groups include, but are not limited to, methyl,ethyl, propyl, alkoxy groups such as —O—(CH₂)₂CH₃ and —O—(CH₂)₄CH₃ andethylene glycol and derivatives thereof.

Preferred aromatic groups include, but are not limited to, phenyl,naphthyl, naphthalene, anthracene, phenanthroline, pyrole, pyridine,thiophene, porphyrins, and substituted derivatives of each of these,included fused ring derivatives.

In the conductive oligomers depicted herein, when g is 1, B-D is a bondlinking two atoms or chemical moieties. In a preferred embodiment, B-Dis a conjugated bond, containing overlapping or conjugated n-orbitals.

Preferred B-D bonds are selected from acetylene (—C≡C—, also calledalkyne or ethyne), alkene (—CH═CH—, also called ethylene), substitutedalkene (—CR═CR—, —CH═CR— and —CR═CH—), amide (—NH—CO— and —NR—CO— or—CO—NH— and —CO—NR—), azo (—N═N—), esters and thioesters (—CO—O—,—O—CO—, —CS—O— and —O—CS—) and other conjugated bonds such as (—CH═N—,—CR═N—, —N═CH— and —N═CR—), (—SiH═SiH—, —SiR═SiH—, —SiR═SiH—, and—SiR═SiR—), (—SiH═CH—, —SiR═CH—, —SiH═CR—, —SiR═CR—, —CH═SiH—, —CR═SiH—,—CH═SiR—, and —CR═SiR—). Particularly preferred B-D bonds are acetylene,alkene, amide, and substituted derivatives of these three, and azo.Especially preferred B-D bonds are acetylene, alkene and amide. Theoligomer components attached to double bonds may be in the trans or cisconformation, or mixtures. Thus, either B or D may include carbon,nitrogen or silicon. The substitution groups are as defined as above forR.

When g=0 in the Structure 1 conductive oligomer, e is preferably 1 andthe D moiety may be carbonyl or a heteroatom moiety as defined above.

As above for the Y rings, within any single conductive oligomer, the B-Dbonds (or D moieties, when g=0) may be all the same, or at least one maybe different. For example, when m is zero, the terminal B-D bond may bean amide bond, and the rest of the B-D bonds may be acetylene bonds.Generally, when amide bonds are present, as few amide bonds as possibleare preferable, but in some embodiments all the B-D bonds are amidebonds. Thus, as outlined above for the Y rings, one type of B-D bond maybe present in the conductive oligomer within a monolayer as describedbelow, and another type above the monolayer level, for example to givegreater flexibility for nucleic acid hybridization when the nucleic acidis attached via a conductive oligomer.

In the structures depicted herein, n is an integer from 1 to 50,although longer oligomers may also be used (see for example Schumm etal., Angew. Chem. Int. Ed. Engl. 1994 33(13):1360). Without being boundby theory, it appears that for efficient hybridization of nucleic acidson a surface, the hybridization should occur at a distance from thesurface, i.e. the kinetics of hybridization increase as a function ofthe distance from the surface, particularly for long oligonucleotides of200 to 300 basepairs. Accordingly, when a nucleic acid is attached via aconductive oligomer, as is more fully described below, the length of theconductive oligomer is such that the closest nucleotide of the nucleicacid is positioned from about 6 Å to about 100 Å (although distances ofup to 500 Å may be used) from the electrode surface, with from about 15Å to about 60 Å being preferred and from about 25 Å to about 60 Å alsobeing preferred. Accordingly, n will depend on the size of the aromaticgroup, but generally will be from about 1 to about 20, with from about 2to about 15 being preferred and from about 3 to about 10 beingespecially preferred.

In the structures depicted herein, m is either 0 or 1. That is, when mis 0, the conductive oligomer may terminate in the B-D bond or D moiety,i.e. the D atom is attached to the nucleic acid either directly or via alinker. In some embodiments, for example when the conductive oligomer isattached to a phosphate of the ribose-phosphate backbone of a nucleicacid, there may be additional atoms, such as a linker, attached betweenthe conductive oligomer and the nucleic acid. Additionally, as outlinedbelow, the D atom may be the nitrogen atom of the amino-modified ribose.Alternatively, when m is 1, the conductive oligomer may terminate in Y,an aromatic group, i.e. the aromatic group is attached to the nucleicacid or linker.

As will be appreciated by those in the art, a large number of possibleconductive oligomers may be utilized. These include conductive oligomersfalling within the Structure 1 and Structure 8 formulas, as well asother conductive oligomers, as are generally known in the art, includingfor example, compounds comprising fused aromatic rings or Teflon®-likeoligomers, such as —(CF₂)_(n)—, —(CHF)_(n)—and —(CFR)_(n)—. See forexample, Schumm et al., Angew. Chem. Intl. Ed. Engl. 33:1361 (1994);Grosshenny et al., Platinum Metals Rev. 40(1):26-35 (1996); Tour, Chem.Rev. 96:537-553 (1996); Hsung et al., Organometallics 14:4808-4815(1995; and references cited therein, all of which are expresslyincorporated by reference.

Particularly preferred conductive oligomers of this embodiment aredepicted below:

Structure 2 is Structure 1 when g is 1. Preferred embodiments ofStructure 2 include: e is zero, Y is pyrole or substituted pyrole; e iszero, Y is thiophene or substituted thiophene; e is zero, Y is furan orsubstituted furan; e is zero, Y is phenyl or substituted phenyl; e iszero, Y is pyridine or substituted pyridine; e is 1, B-D is acetyleneand Y is phenyl or substituted phenyl (see Structure 4 below). Apreferred embodiment of Structure 2 is also when e is one, depicted asStructure 3 below:

Preferred embodiments of Structure 3 are: Y is phenyl or substitutedphenyl and B-D is azo; Y is phenyl or substituted phenyl and B-D isacetylene; Y is phenyl or substituted phenyl and B-D is alkene; Y ispyridine or substituted pyridine and B-D is acetylene; Y is thiophene orsubstituted thiophene and B-D is acetylene; Y is furan or substitutedfuran and B-D is acetylene; Y is thiophene or furan (or substitutedthiophene or furan) and B-D are alternating alkene and acetylene bonds.

Most of the structures depicted herein utilize a Structure 3 conductiveoligomer. However, any Structure 3 oligomers may be substituted with anyof the other structures depicted herein, i.e. Structure 1 or 8 oligomer,or other conducting oligomer, and the use of such Structure 3 depictionis not meant to limit the scope of the invention.

Particularly preferred embodiments of Structure 3 include Structures 4,5, 6 and 7, depicted below:

Particularly preferred embodiments of Structure 4 include: n is two, mis one, and R is hydrogen; n is three, m is zero, and R is hydrogen; andthe use of R groups to increase solubility.

When the B-D bond is an amide bond, as in Structure 5, the conductiveoligomers are pseudopeptide oligomers. Although the amide bond inStructure 5 is depicted with the carbonyl to the left, i.e. —CONN—, thereverse may also be used, i.e. —NHCO—. Particularly preferredembodiments of Structure 5 include: n is two, m is one, and R ishydrogen; n is three, m is zero, and R is hydrogen (in this embodiment,the terminal nitrogen (the D atom) may be the nitrogen of theamino-modified ribose); and the use of R groups to increase solubility.

Preferred embodiments of Structure 6 include the first n is two, secondn is one, m is zero, and all R groups are hydrogen, or the use of Rgroups to increase solubility.

Preferred embodiments of Structure 7 include: the first n is three, thesecond n is from 1-3, with m being either 0 or 1, and the use of Rgroups to increase solubility.

In a preferred embodiment, the conductive oligomer has the structuredepicted in Structure 8:

In this embodiment, C are carbon atoms, n is an integer from 1 to 50, mis 0 or 1, J is a heteroatom selected from the group consisting ofoxygen, nitrogen, silicon, phosphorus, sulfur, carbonyl or sulfoxide,and G is a bond selected from alkane, alkene or acetylene, such thattogether with the two carbon atoms the C-G-C group is an alkene(—CH═CH—), substituted alkene (—CR═CR—) or mixtures thereof (—CH═CR— or—CR═CH—), acetylene (—C≡C—), or alkane (—CR₂—CR₂—, with R being eitherhydrogen or a substitution group as described herein). The G bond ofeach subunit may be the same or different than the G bonds of othersubunits; that is, alternating oligomers of alkene and acetylene bondscould be used, etc. However, when G is an alkane bond, the number ofalkane bonds in the oligomer should be kept to a minimum, with about sixor less sigma, bonds per conductive oligomer being preferred. Alkenebonds are preferred, and are generally depicted herein, although alkaneand acetylene bonds may be substituted in any structure or embodimentdescribed herein as will be appreciated by those in the art.

In some embodiments, for example when ETMs are not present, if m=0 thenat least one of the G bonds is not an alkane bond.

In a preferred embodiment, the m of Structure 8 is zero. In aparticularly preferred embodiment, m is zero and G is an alkene bond, asis depicted in Structure 9:

The alkene oligomer of structure 9, and others depicted herein, aregenerally depicted in the preferred trans configuration, althougholigomers of cis or mixtures of trans and cis may also be used. Asabove, R groups may be added to alter the packing of the compositions onan electrode, the hydrophilicity or hydrophobicity of the oligomer, andthe flexibility, i.e. the rotational, torsional or longitudinal of theoligomer. n is as defined above.

In a preferred embodiment, R is hydrogen, although R may be also alkylgroups and polyethylene glycols or derivatives.

In an alternative embodiment, the conductive oligomer may be a mixtureof different types of oligomers, for example of structures 1 and 8.

In addition, the terminus of at least some of the conductive oligomersin the monolayer are electronically exposed. By “electronically exposed”herein is meant that upon the placement of an ETM in close proximity tothe terminus, and after initiation with the appropriate signal, a signaldependent on the presence of the ETM may be detected. The conductiveoligomers may or may not have terminal groups. Thus, in a preferredembodiment, there is no additional terminal group, and the conductiveoligomer terminates with one of the groups depicted in Structures 1 to9; for example, a B-D bond such as an acetylene bond. Alternatively, ina preferred embodiment, a terminal group is added, sometimes depictedherein as “Q”. A terminal group may be used for several reasons; forexample, to contribute to the electronic availability of the conductiveoligomer for detection of ETMs, or to alter the surface of the SAM forother reasons, for example to prevent non-specific binding. For example,there may be negatively charged groups on the terminus to form anegatively charged surface such that when the nucleic acid is DNA or RNAthe nucleic acid is repelled or prevented from lying down on thesurface, to facilitate hybridization. Preferred terminal groups include—NH₂, —OH, —COOH, and alkyl groups such as —CH₃, and (poly)alkyloxidessuch as (poly)ethylene glycol, with —OCH₂CH₂OH, —(OCH₂CH₂O)₂H,—(OCH₂CH₂O)₃H, and —(OCH₂CH₂O)₄H being preferred.

In one embodiment, it is possible to use mixtures of conductiveoligomers with different types of terminal groups. Thus, for example,some of the terminal groups may facilitate detection, and some mayprevent non-specific binding.

It will be appreciated that the monolayer may comprise differentconductive oligomer species, although preferably the different speciesare chosen such that a reasonably uniform SAM can be formed. Thus, forexample, when nucleic acids are covalently attached to the electrodeusing conductive oligomers, it is possible to have one type ofconductive oligomer used to attach the nucleic acid, and another typefunctioning to detect the ETM. Similarly, it may be desirable to havemixtures of different lengths of conductive oligomers in the monolayer,to help reduce non-specific signals. Thus, for example, preferredembodiments utilize conductive oligomers that terminate below thesurface of the rest of the monolayer, i.e. below the insulator layer, ifused, or below some fraction of the other conductive oligomers.Similarly, the use of different conductive oligomers may be done tofacilitate monolayer formation, or to make monolayers with alteredproperties.

In a preferred embodiment, the monolayer forming species are“interrupted” conductive oligomers, containing an alkyl portion in themiddle of the conductive oligomer.

In a preferred embodiment, the monolayer comprises photoactivatablespecies as EFS. Photoactivatable species are known in the art, andinclude 4,5-dimethoxy-2-nitrobenzyl ester, which can be photolyzed at365 nm for 2 hours.

In a preferred embodiment, the monolayer may further comprise insulatormoieties. By “insulator” herein is meant a substantially nonconductingoligomer, preferably linear. By “substantially nonconducting” herein ismeant that the insulator will not transfer electrons at 100 Hz. The rateof electron transfer through the insulator is preferrably slower thanthe rate through the conductive oligomers described herein.

In a preferred embodiment, the insulators have a conductivity, S, ofabout 10⁻⁷ Ω⁻¹·cm⁻¹ or lower, with less than about 10⁻⁸ Ω⁻¹·cm⁻¹ beingpreferred. See generally Gardner et al., supra.

Generally, insulators are alkyl or heteroalkyl oligomers or moietieswith sigma bonds, although any particular insulator molecule may containaromatic groups or one or more conjugated bonds. By “heteroalkyl” hereinis meant an alkyl group that has at least one heteroatom, i.e. nitrogen,oxygen, sulfur, phosphorus, silicon or boron included in the chain.Alternatively, the insulator may be quite similar to a conductiveoligomer with the addition of one or more heteroatoms or bonds thatserve to inhibit or slow, preferably substantially, electron transfer.

Suitable insulators are known in the art, and include, but are notlimited to, —(CH₂)_(n)—, —(CRH)_(n)—, and —(CR₂)_(n)—, ethylene glycolor derivatives using other heteroatoms in place of oxygen, i.e. nitrogenor sulfur (sulfur derivatives are not preferred when the electrode isgold).

As for the conductive oligomers, the insulators may be substituted withR groups as defined herein to alter the packing of the moieties orconductive oligomers on an electrode, the hydrophilicity orhydrophobicity of the insulator, and the flexibility, i.e. therotational, torsional or longitudinal flexibility of the insulator. Forexample, branched alkyl groups may be used. Similarly, the insulatorsmay contain terminal groups, as outlined above, particularly toinfluence the surface of the monolayer.

The length of the species making up the monolayer will vary as needed.As outlined above, it appears that hybridization is more efficient at adistance from the surface. The species to which nucleic acids areattached (as outlined below, these can be either insulators orconductive oligomers) may be basically the same length as the monolayerforming species or longer than them, resulting in the nucleic acidsbeing more accessible to the solvent for hybridization. In someembodiments, the conductive oligomers to which the nucleic acids areattached may be shorter than the monolayer.

As will be appreciated by those in the art, the actual combinations andratios of the different species making up the monolayer can vary widely,and will depend on whether mechanism-1 or -2 is used. Generally, threecomponent systems are preferred for mechanism-2 systems, with the firstspecies comprising a capture probe containing species, attached to theelectrode via either an insulator or a conductive oligomer. The secondspecies are EFS, preferably conductive oligomers, and the third speciesare insulators. In this embodiment, the first species can comprise fromabout 90% to about 1%, with from about 20% to about 40% being preferred.For nucleic acids, from about 30% to about 40% is especially preferredfor short oligonucleotide targets and from about 10% to about 20% ispreferred for longer targets. The second species can comprise from about1% to about 90%, with from about 20% to about 90% being preferred, andfrom about 40% to about 60% being especially preferred. The thirdspecies can comprise from about 1% to about 90%, with from about 20% toabout 40% being preferred, and from about 15% to about 30% beingespecially preferred. To achieve these approximate proportions,preferred ratios of first:second:third species in SAM formation solventsare 2:2:1 for short targets, 1:3:1 for longer targets, with total thiolconcentration (when used to attach these species, as is more fullyoutlined below) in the 500 μM to 1 mM range, and 833 μM being preferred.

Alternatively, two component systems can be used. In one embodiment, foruse in either mechanism-1 or mechanism-2 systems, the two components arethe first and second species. In this embodiment, the first species cancomprise from about 1% to about 90%, with from about 1% to about 40%being preferred, and from about 10% to about 40% being especiallypreferred. The second species can comprise from about 1% to about 90%,with from about 10% to about 60% being preferred, and from about 20% toabout 40% being especially preferred. Alternatively, for mechanism-1systems, the two components are the first and the third species. In thisembodiment, the first species can comprise from about 1% to about 90%,with from about 1% to about 40% being preferred, and from about 10% toabout 40% being especially preferred. The second species can comprisefrom about 1% to about 90%, with from about 10% to about 60% beingpreferred, and from about 20% to about 40% being especially preferred.

In a preferred embodiment, the deposition of the SAM is done usingaqueous solvents. As is generally described in Steel et al., Anal. Chem.70:4670 (1998), Herne et al., J. Am. Chem. Soc. 119:8916 (1997), andFinklea, Electrochemistry of Organized Monolayers of Thiols and RelatedMolecules on Electrodes, from A. J. Bard, Electroanalytical Chemistry: ASeries of Advances, Vol. 20, Dekker N.Y. 1966-, all of which areexpressly incorporated by reference, the deposition of the SAM-formingspecies can be done out of aqueous solutions, frequently comprisingsalt.

The covalent attachment of the conductive oligomers and insulators maybe accomplished in a variety of ways, depending on the electrode and thecomposition of the insulators and conductive oligomers used. In apreferred embodiment, the attachment linkers with covalently attachedcapture binding ligands (e.g. nucleosides or nucleic acids) as depictedherein are covalently attached to an electrode. Thus, one end orterminus of the attachment linker is attached to the nucleoside ornucleic acid, and the other is attached to an electrode. In someembodiments it may be desirable to have the attachment linker attachedat a position other than a terminus, or even to have a branchedattachment linker that is attached to an electrode at one terminus andto two or more nucleosides at other termini, although this is notpreferred. Similarly, the attachment linker may be attached at two sitesto the electrode, as is generally depicted in Structures 11-13.Generally, some type of linker is used, as depicted below as “A” inStructure 10, where “X” is the conductive oligomer, “I” is an insulatorand the hatched surface is the electrode:

In this embodiment, A is a linker or atom. The choice of “A” will dependin part on the characteristics of the electrode. Thus, for example, Amay be a sulfur moiety when a gold electrode is used. Alternatively,when metal oxide electrodes are used, A may be a silicon (silane) moietyattached to the oxygen of the oxide (see for example Chen et al.Langmuir 10:3332-3337 (1994); Lenhard et al., J. Electroanal. Chem.78:195-201 (1977), both of which are expressly incorporated byreference). When carbon based electrodes are used, A may be an aminomoiety (preferably a primary amine; see for example Deinhammer et al.,Langmuir 10:1306-1313 (1994)). Thus, preferred A moieties include, butare not limited to, silane moieties, sulfur moieties (including alkylsulfur moieties), and amino moieties. In a preferred embodiment, epoxidetype linkages with redox polymers such as are known in the art are notused.

Although depicted herein as a single moiety, the insulators andconductive oligomers may be attached to the electrode with more than one“A” moiety; the “A” moieties may be the same or different. Thus, forexample, when the electrode is a gold electrode, and “A” is a sulfuratom or moiety, multiple sulfur atoms may be used to attach theconductive oligomer to the electrode, such as is generally depictedbelow in Structures 11, 12 and 13. As will be appreciated by those inthe art, other such structures can be made. In Structures 11, 12 and 13,the A moiety is just a sulfur atom, but substituted sulfur moieties mayalso be used.

It should also be noted that similar to Structure 13, it may be possibleto have a a conductive oligomer terminating in a single carbon atom withthree sulfur moities attached to the electrode. Additionally, althoughnot always depicted herein, the conductive oligomers and insulators mayalso comprise a “Q” terminal group.

In a preferred embodiment, the electrode is a gold electrode, andattachment is via a sulfur linkage as is well known in the art, i.e. theA moiety is a sulfur atom or moiety. Although the exact characteristicsof the gold-sulfur attachment are not known, this linkage is consideredcovalent for the purposes of this invention. A representative structureis depicted in Structure 14, using the Structure 3 conductive oligomer,although as for all the structures depicted herein, any of theconductive oligomers, or combinations of conductive oligomers, may beused. Similarly, any of the conductive oligomers or insulators may alsocomprise terminal groups as described herein. Structure 14 depicts the“A” linker as comprising just a sulfur atom, although additional atomsmay be present (i.e. linkers from the sulfur to the conductive oligomeror substitution groups). In addition, Structure 14 shows the sulfur atomattached to the Y aromatic group, but as will be appreciated by those inthe art, it may be attached to the B-D group (i.e. an acetylene) aswell.

In a preferred embodiment, the electrode is a carbon electrode, i.e. aglassy carbon electrode, and attachment is via a nitrogen of an aminegroup. A representative structure is depicted in Structure 15. Again,additional atoms may be present, i.e. Z type linkers and/or terminalgroups.

In Structure 16, the oxygen atom is from the oxide of the metal oxideelectrode. The Si atom may also contain other atoms, i.e. be a siliconmoiety containing substitution groups. Other attachments for SAMs toother electrodes are known in the art; see for example Napier et al.,Langmuir, 1997, for attachment to indium tin oxide electrodes, and alsothe chemisorption of phosphates to an indium tin oxide electrode (talkby H. Holden Thorpe, CHI conference, May 4-5, 1998).

The SAMs of the invention can be made in a variety of ways, includingdeposition out of organic solutions and deposition out of aqueoussolutions. The methods outlined herein use a gold electrode as theexample, although as will be appreciated by those in the art, othermetals and methods may be used as well. In one preferred embodiment,indium-tin-oxide (ITO) is used as the electrode.

In a preferred embodiment, a gold surface is first cleaned. A variety ofcleaning procedures may be employed, including, but not limited to,chemical cleaning or etchants (including Piranha solution (hydrogenperoxide/sulfuric acid) or aqua regia (hydrochloric acid/nitric acid),electrochemical methods, flame treatment, plasma treatment orcombinations thereof.

Following cleaning, the gold substrate is exposed to the SAM species.When the electrode is ITO, the SAM species are phosphonate-containingspecies. This can also be done in a variety of ways, including, but notlimited to, solution deposition, gas phase deposition, microcontactprinting, spray deposition, deposition using neat components, etc. Apreferred embodiment utilizes a deposition solution comprising a mixtureof various SAM species in solution, generally thiol-containing species.Mixed monolayers that contain nucleic acids are usually prepared using atwo step procedure. The thiolated nucleic acid is deposited during thefirst deposition step (generally in the presence of at least one othermonolayer-forming species) and the mixed monolayer formation iscompleted during the second step in which a second thiol solution minusnucleic acid is added. The second step frequently involves mild heatingto promote monolayer reorganization.

In a preferred embodiment, the deposition solution is an organicdeposition solution. In this embodiment, a clean gold surface is placedinto a clean vial. A binding ligand deposition solution in organicsolvent is prepared in which the total thiol concentration is betweenmicromolar to saturation; preferred ranges include from about 1 μM to 10mM, with from about 400 uM to about 1.0 mM being especially preferred.In a preferred embodiment, the deposition solution contains thiolmodified DNA (i.e. nucleic acid attached to an attachment linker) andthiol diluent molecules (either conductive oligomers or insulators, withthe latter being preferred). The ratio of nucleic acid to diluent (ifpresent) is usually between 1000:1 to 1:1000, with from about 10:1 toabout 1:10 being preferred and 1:1 being especially preferred. Thepreferred solvents are tetrahydrofuran (THF), acetonitrile,dimethylforamide (DMF), ethanol, or mixtures thereof; generally anysolvent of sufficient polarity to dissolve the capture ligand can beused, as long as the solvent is devoid of functional groups that willreact with the surface. Sufficient nucleic acid deposition solution isadded to the vial so as to completely cover the electrode surface. Thegold substrate is allowed to incubate at ambient temperature or slightlyabove ambient temperature for a period of time ranging from seconds tohours, with 5-30 minutes being preferred. After the initial incubation,the deposition solution is removed and a solution of diluent moleculeonly (from about 1 μM to 10 mM, with from about 100 uM to about 1.0 mMbeing preferred) in organic solvent is added. The gold substrate isallowed to incubate at room temperature or above room temperature for aperiod of time (seconds to days, with from about 10 minutes to about 24hours being preferred). The gold sample is removed from the solution,rinsed in clean solvent and used.

In a preferred embodiment, an aqueous deposition solution is used. Asabove, a clean gold surface is placed into a clean vial. A nucleic aciddeposition solution in water is prepared in which the total thiolconcentration is between about 1 uM and 10 mM, with from about 1 μM toabout 200 uM being preferred. The aqueous solution frequently has saltpresent (up to saturation, with approximately 1M being preferred),however pure water can be used. The deposition solution contains thiolmodified nucleic acid and often a thiol diluent molecule. The ratio ofnucleic acid to diluent is usually between between 1000:1 to 1:1000,with from about 10:1 to about 1:10 being preferred and 1:1 beingespecially preferred. The nucleic acid deposition solution is added tothe vial in such a volume so as to completely cover the electrodesurface. The gold substrate is allowed to incubate at ambienttemperature or slightly above ambient temperature for 1-30 minutes with5 minutes usually being sufficient. After the initial incubation, thedeposition solution is removed and a solution of diluent molecule only(10 uM-1.0 mM) in either water or organic solvent is added. The goldsubstrate is allowed to incubate at room temperature or above roomtemperature until a complete monolayer is formed (10 minutes-24 hours).The gold sample is removed from the solution, rinsed in clean solventand used.

In a preferred embodiment, as outlined herein, a circuit board is usedas the substrate for the gold electrodes. Formation of the SAMs on thegold surface is generally done by first cleaning the boards, for examplein a 10% sulfuric acid solution for 30 seconds, detergent solutions,aqua regia, plasma, etc., as outlined herein. Following the sulfuricacid treatment, the boards are washed, for example via immersion in twoMilli-Q water baths for 1 minute each. The boards are then dried, forexample under a stream of nitrogen. Spotting of the deposition solutiononto the boards is done using any number of known spotting systems,generally by placing the boards on an X-Y table, preferably in ahumidity chamber. The size of the spotting drop will vary with the sizeof the electrodes on the boards and the equipment used for delivery ofthe solution; for example, for 250 μM size electrodes, a 30 nanoliterdrop is used. The volume should be sufficient to cover the electrodesurface completely. The drop is incubated at room temperature for aperiod of time (sec to overnight, with 5 minutes preferred) and then thedrop is removed by rinsing in a Milli-Q water bath. The boards are thenpreferably treated with a second deposition solution, generallycomprising insulator in organic solvent, preferably acetonitrile, byimmersion in a 45° C. bath. After 30 minutes, the boards are removed andimmersed in an acetonitrile bath for 30 seconds followed by a milli-Qwater bath for 30 seconds. The boards are dried under a stream ofnitrogen.

In a preferred embodiment, the electrode comprising the monolayerfurther comprises a capture binding ligand covalently attached to theelectrode. This attachment can be via an attachment linker, which may bea conductive oligomer or an insulator. By “capture binding ligand” or“capture binding species” or “capture probe” herein is meant a compoundthat is used to probe for the presence of the target analyte, that willbind to the target analyte. (“Capture probe” or “anchor probe” areparticularly used when the capture binding ligand is a nucleic acid).Generally, the capture binding ligand allows the attachment of a targetanalyte to the electrode, for the purposes of detection. As is morefully outlined below, attachment of the target analyte to the captureprobe may be direct (i.e. the target analyte binds to the capturebinding ligand) or indirect (one or more capture extender ligands areused). By “covalently attached” herein is meant that two moieties areattached by at least one bond, including sigma bonds, pi bonds andcoordination bonds.

In a preferred embodiment, the binding is specific, and the bindingligand is part of a binding pair. By “specifically bind” herein is meantthat the ligand binds the analyte, with specificity sufficient todifferentiate between the analyte and other components or contaminantsof the test sample. However, as will be appreciated by those in the art,it will be possible to detect analytes using binding which is not highlyspecific; for example, the systems may use different binding ligands,for example an array of different ligands, and detection of anyparticular analyte is via its “signature” of binding to a panel ofbinding ligands, similar to the manner in which “electronic noses” work.This finds particular utility in the detection of chemical analytes. Thebinding should be sufficient to remain bound under the conditions of theassay, including wash steps to remove non-specific binding. In someembodiments, for example in the detection of certain biomolecules, thebinding constants of the analyte to the binding ligand will be at leastabout 10⁴-10⁶ M⁻¹, with at least about 10⁵ to 10⁹M⁻¹ being preferred andat least about 10⁷-10⁹ M⁻¹ being particularly preferred.

As will be appreciated by those in the art, the composition of thebinding ligand will depend on the composition of the target analyte.Binding ligands to a wide variety of analytes are known or can bereadily found using known techniques. For example, when the analyte is asingle-stranded nucleic acid, the binding ligand may be a complementarynucleic acid. Similarly, the analyte may be a nucleic acid bindingprotein and the capture binding ligand is either single-stranded ordouble stranded nucleic acid; alternatively, the binding ligand may be anucleic acid-binding protein when the analyte is a single ordouble-stranded nucleic acid. When the analyte is a protein, the bindingligands include proteins or small molecules. Preferred binding ligandproteins include peptides. For example, when the analyte is an enzyme,suitable binding ligands include substrates and inhibitors. As will beappreciated by those in the art, any two molecules that will associatemay be used, either as an analyte or as the binding ligand. Suitableanalyte/binding ligand pairs include, but are not limited to,antibodies/antigens, receptors/ligands, proteins/nucleic acid,enzymes/substrates and/or inhibitors, carbohydrates (includingglycoproteins and glycolipids)/lectins, proteins/proteins,proteins/small molecules; and carbohydrates and their binding partnersare also suitable analyte-binding ligand pairs. These may be wild-typeor derivative sequences. In a preferred embodiment, the binding ligandsare portions (particularly the extracellular portions) of cell surfacereceptors that are known to multimerize, such as the growth hormonereceptor, glucose transporters (particularly GLUT 4 receptor),transferrin receptor, epidermal growth factor receptor, low densitylipoprotein receptor, high density lipoprotein receptor, epidermalgrowth factor receptor, leptin receptor, interleukin receptors includingIL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-11, IL-12,IL-13, IL-15, and IL-17 receptors, human growth hormone receptor, VEGFreceptor, PDGF receptor, EPO receptor, TPO receptor, ciliaryneurotrophic factor receptor, prolactin receptor, and T-cell receptors.

The method of attachment of the capture binding ligand to the attachmentlinker will generally be done as is known in the art, and will depend onthe composition of the attachment linker and the capture binding ligand.In general, the capture binding ligands are attached to the attachmentlinker through the use of functional groups on each that can then beused for attachment. Preferred functional groups for attachment areamino groups, carboxy groups, oxo groups and thiol groups. Thesefunctional groups can then be attached, either directly or through theuse of a linker, sometimes depicted herein as “Z”. Linkers are known inthe art; for example, homo- or hetero-bifunctional linkers as are wellknown (see 1994 Pierce Chemical Company catalog, technical section oncross-linkers, pages 155-200, incorporated herein by reference).Preferred Z linkers include, but are not limited to alkyl groups(including substituted alkyl groups and alkyl groups containingheteroatom moieties), with short alkyl groups, esters, amide, amine,epoxy groups and ethylene glycol and derivatives being preferred. Z mayalso be a sulfone group, forming sulfonamide.

In this way, capture binding ligands comprising proteins, lectins,nucleic acids, small organic molecules, carbohydrates, etc. can beadded.

In a preferred embodiment, the capture binding ligand is attacheddirectly to the electrode as outlined herein, for example via anattachment linker. Alternatively, the capture binding ligand may utilizea capture extender component. In this embodiment, the capture bindingligand comprises a first portion that will bind the target analyte and asecond portion that can be used for attachment to the surface. FIG. 2Cdepicts the use of a nucleic acid component for binding to the surface,although this can be other binding partners as well.

A preferred embodiment utilizes proteinaceous capture binding ligands.As is known in the art, any number of techniques may be used to attach aproteinaceous capture binding ligand. “Protein” in this context includesproteins, polypeptides and peptides. A wide variety of techniques areknown to add moieties to proteins. One preferred method is outlined inU.S. Pat. No. 5,620,850, hereby incorporated by reference in itsentirety. The attachment of proteins to electrodes is known; see alsoHeller, Acc. Chem. Res. 23:128 (1990), and related work.

A preferred embodiment utilizes nucleic acids as the capture bindingligand, for example for when the target analyte is a nucleic acid or anucleic acid binding protein, or when the nucleic acid serves as anaptamer for binding a protein; see U.S. Pat. Nos. 5,270,163, 5,475,096,5,567,588, 5,595,877, 5,637,459, 5,683,867, 5,705,337, and relatedpatents, hereby incorporated by reference. In this embodiment, thenucleic acid capture binding ligand is covalently attached to theelectrode, via an “attachment linker”, that can be either a conductiveoligomer or via an insulator. Thus, one end of the attachment linker isattached to a nucleic acid, and the other end (although as will beappreciated by those in the art, it need not be the exact terminus foreither) is attached to the electrode.

As is more fully outlined below, attachment of the target sequence tothe capture probe may be direct (i.e. the target sequence hybridizes tothe capture probe) or indirect (one or more capture extender probes areused). In addition, as is more fully outlined below, the capture probesmay have both nucleic and non-nucleic acid portions. Thus, for example,flexible linkers such as alkyl groups, including polyethylene glycollinkers, may be used to get the nucleic acid portion of the captureprobe off the electrode surface. This may be particularly useful whenthe target sequences are large, for example when genomic DNA or rRNA isthe target.

The capture probe nucleic acid is covalently attached to the electrode,via an “attachment linker”, that can be either a conductive oligomer orvia an insulator. Thus, one end of the attachment linker is attached toa nucleic acid, and the other end (although as will be appreciated bythose in the art, it need not be the exact terminus for either) isattached to the electrode. Thus, any of structures depicted herein mayfurther comprise a nucleic acid effectively as a terminal group. Thus,the present invention provides compositions comprising nucleic acidscovalently attached to electrodes as is generally depicted below inStructure 17:

In Structure 17, the hatched marks on the left represent an electrode. Xis a conductive oligomer and I is an insulator as defined herein. F₁ isa linkage that allows the covalent attachment of the electrode and theconductive oligomer or insulator, including bonds, atoms or linkers suchas is described herein, for example as “A”, defined below. F₂ is alinkage that allows the covalent attachment of the conductive oligomeror insulator to the nucleic acid, and may be a bond, an atom or alinkage as is herein described. F₂ may be part of the conductiveoligomer, part of the insulator, part of the nucleic acid, or exogeneousto both, for example, as defined herein for “Z”.

In a preferred embodiment, the capture probe nucleic acid is covalentlyattached to the electrode via a conductive oligomer. The covalentattachment of the nucleic acid and the conductive oligomer may beaccomplished in several ways. In a preferred embodiment, the attachmentis via attachment to the base of the nucleoside, via attachment to thebackbone of the nucleic acid (either the ribose, the phosphate, or to ananalogous group of a nucleic acid analog backbone), or via a transitionmetal ligand, as described below. The techniques outlined below aregenerally described for naturally occurring nucleic acids, although aswill be appreciated by those in the art, similar techniques may be usedwith nucleic acid analogs.

In a preferred embodiment, the conductive oligomer is attached to thebase of a nucleoside of the nucleic acid. This may be done in severalways, depending on the oligomer, as is described below. In oneembodiment, the oligomer is attached to a terminal nucleoside, i.e.either the 3′ or 5′ nucleoside of the nucleic acid. Alternatively, theconductive oligomer is attached to an internal nucleoside.

The point of attachment to the base will vary with the base. Generally,attachment at any position is possible. In some embodiments, for examplewhen the probe containing the ETMs may be used for hybridization, it ispreferred to attach at positions not involved in hydrogen bonding to thecomplementary base. Thus, for example, generally attachment is to the 5or 6 position of pyrimidines such as uridine, cytosine and thymine. Forpurines such as adenine and guanine, the linkage is preferably via the 8position. Attachment to non-standard bases is preferably done at thecomparable positions.

In one embodiment, the attachment is direct; that is, there are nointervening atoms between the conductive oligomer and the base. In thisembodiment, for example, conductive oligomers with terminal acetylenebonds are attached directly to the base. Structure 18 is an example ofthis linkage, using a Structure 3 conductive oligomer and uridine as thebase, although other bases and conductive oligomers can be used as willbe appreciated by those in the art.

It should be noted that the pentose structures depicted herein may havehydrogen, hydroxy, phosphates or other groups such as amino groupsattached. In addition, the pentose and nucleoside structures depictedherein are depicted non-conventionally, as mirror images of the normalrendering. In addition, the pentose and nucleoside structures may alsocontain additional groups, such as protecting, groups, at any position,for example as needed during synthesis.

In addition, the base may contain additional modifications as needed,i.e. the carbonyl or amine groups may be altered or protected, forexample as is depicted in FIG. 18A of PCT US97/20014. This may berequired to prevent significant dimerization of conductive oligomersinstead of coupling to the iodinating base. In addition, changing thecomponents of the palladium reaction may be desirable also. R groups maybe preferred on longer conductive oligomers to increase solubility.

In an alternative embodiment, the attachment is any number of differentZ linkers, including amide and amine linkages, as is generally depictedin Structure 19 using uridine as the base and a Structure 3 oligomer:

In this embodiment, Z is a linker. Preferably, Z is a short linker ofabout 1 to about 10 atoms, with from 1 to 5 atoms being preferred; thatmay or may not contain alkene, alkynyl, amine, amide, azo, imine, etc.,bonds. Linkers are known in the art; for example, homo- orhetero-bifunctional linkers as are well known (see 1994 Pierce ChemicalCompany catalog, technical section on cross-linkers, pages 155-200,incorporated herein by reference). Preferred Z linkers include, but arenot limited to, alkyl groups (including substituted alkyl groups andalkyl groups containing heteroatom moieties), with short alkyl groups,esters, amide, amine, epoxy groups and ethylene glycol and derivativesbeing preferred, with propyl, acetylene, and C₂ alkene being especiallypreferred. Z may also be a sulfone group, forming sulfonamide linkagesas discussed below.

In a preferred embodiment, the attachment of the nucleic acid and theconductive oligomer is done via attachment to the backbone of thenucleic acid. This may be done in a number of ways, including attachmentto a ribose of the ribose-phosphate backbone, or to the phosphate of thebackbone, or other groups of analogous backbones.

As a preliminary matter, it should be understood that the site ofattachment in this embodiment may be to a 3′ or 5′ terminal nucleotide,or to an internal nucleotide, as is more fully described below.

In a preferred embodiment, the conductive oligomer is attached to theribose of the ribose-phosphate backbone. This may be done in severalways. As is known in the art, nucleosides that are modified at eitherthe 2′ or 3′ position of the ribose with amino groups, sulfur groups,silicone groups, phosphorus groups, or oxo groups can be made (Imazawaet al., J. Org. Chem., 44:2039 (1979); Hobbs et al., J. Org. Chem.42(4):714 (1977); Verheyden et al., J. Orrg. Chem. 36(2):250 (1971);McGee et al., J. Org. Chem. 61:781-785 (1996); Mikhailopulo et al.,Liebigs. Ann. Chem. 513-519 (1993); McGee et al., Nucleosides &Nucleotides 14(6):1329 (1995), all of which are incorporated byreference). These modified nucleosides are then used to add theconductive oligomers.

A preferred embodiment utilizes amino-modified nucleosides. Theseamino-modified riboses can then be used to form either amide or aminelinkages to the conductive oligomers. In a preferred embodiment, theamino group is attached directly to the ribose, although as will beappreciated by those in the art, short linkers such as those describedherein for “Z” may be present between the amino group and the ribose.

In a preferred embodiment, an amide linkage is used for attachment tothe ribose. Preferably, if the conductive oligomer of Structures 1-3 isused, m is zero and thus the conductive oligomer terminates in the amidebond. In this embodiment, the nitrogen of the amino group of theamino-modified ribose is the “D” atom of the conductive oligomer. Thus,a preferred attachment of this embodiment is depicted in Structure 20(using the Structure 3 conductive oligomer):

As will be appreciated by those in the art, Structure 20 has theterminal bond fixed as an amide bond.

In a preferred embodiment, a heteroatom linkage is used, i.e. oxo,amine, sulfur, etc. A preferred embodiment utilizes an amine linkage.Again, as outlined above for the amide linkages, for amine linkages, thenitrogen of the amino-modified ribose may be the “D” atom of theconductive oligomer when the Structure 3 conductive oligomer is used.Thus, for example, Structures 21 and 22 depict nucleosides with theStructures. 3 and 9 conductive oligomers, respectively, using thenitrogen as the heteroatom, athough other heteroatoms can be used:

In Structure 21, preferably both m and t are not zero. A preferred Zhere is a methylene group, or other aliphatic alkyl linkers. One, two orthree carbons in this position are particularly useful for syntheticreasons; see PCT US97/20014.

In Structure 22, Z is as defined above. Suitable linkers includemethylene and ethylene.

In an alternative embodiment, the conductive oligomer is covalentlyattached to the nucleic acid via the phosphate of the ribose-phosphatebackbone (or analog) of a nucleic acid. In this embodiment, theattachment is direct, utilizes a linker or via an amide bond. Structure23 depicts a direct linkage, and Structure 24 depicts linkage via anamide bond (both utilize the Structure 3 conductive oligomer, althoughStructure 8 conductive oligomers are also possible). Structures 23 and24 depict the conductive oligomer in the 3′ position, although the 5′position is also possible. Furthermore, both Structures 23 and 24 depictnaturally occurring phosphodiester bonds, although as those in the artwill appreciate, non-standard analogs of phosphodiester bonds may alsobe used.

In Structure 23, if the terminal Y is present (i.e. m=1), thenpreferably Z is not present (i.e. t=0). If the terminal Y is notpresent, then Z is preferably present.

Structure 24 depicts a preferred embodiment, wherein the terminal B-Dbond is an amide bond, the terminal Y is not present, and Z is a linker,as defined herein.

In a preferred embodiment, the conductive oligomer is covalentlyattached to the nucleic acid via a transition metal ligand. In thisembodiment, the conductive oligomer is covalently attached to a ligandwhich provides one or more of the coordination atoms for a transitionmetal. In one embodiment, the ligand to which the conductive oligomer isattached also has the nucleic acid attached, as is generally depictedbelow in Structure 25. Alternatively, the conductive oligomer isattached to one ligand, and the nucleic acid is attached to anotherligand, as is generally depicted below in Structure 26. Thus, in thepresence of the transition metal, the conductive oligomer is covalentlyattached to the nucleic acid. Both of these structures depict Structure3 conductive oligomers, although other oligomers may be utilized.Structures 25 and 26 depict two representative structures:

In the structures depicted herein, M is a metal atom, with transitionmetals being preferred. Suitable transition metals for use in theinvention include, but are not limited to, cadmium (Cd), copper (Cu),cobalt (Co), palladium (Pd), zinc (Zn), iron (Fe), ruthenium (Ru),rhodium (Rh), osmium (Os), rhenium (Re), platinium (Pt), scandium (Sc),titanium (Ti), Vanadium (V), chromium (Cr), manganese (Mn), nickel (Ni),Molybdenum (Mo), technetium (Tc), tungsten (W), and iridium (Ir). Thatis, the first series of transition metals, the platinum metals (Ru, Rh,Pd, Os, Ir and Pt), along with Fe, Re, W, Mo and Tc, are preferred.Particularly preferred are ruthenium, rhenium, osmium, platinium, cobaltand iron.

L are the co-ligands, that provide the coordination atoms for thebinding of the metal ion. As will be appreciated by those in the art,the number and nature of the co-ligands will depend on the coordinationnumber of the metal ion. Mono-, di- or polydentate co-ligands may beused at any position. Thus, for example, when the metal has acoordination number of six, the L from the terminus of the conductiveoligomer, the L contributed from the nucleic acid, and r, add up to six.Thus, when the metal has a coordination number of six, r may range fromzero (when all coordination atoms are provided by the other two ligands)to four, when all the co-ligands are monodentate. Thus generally, r willbe from 0 to 8, depending on the coordination number of the metal ionand the choice of the other ligands.

In one embodiment, the metal ion has a coordination number of six andboth the ligand attached to the conductive oligomer and the ligandattached to the nucleic acid are at least bidentate; that is, r ispreferably zero, one (i.e. the remaining co-ligand is bidentate) or two(two monodentate co-ligands are used).

As will be appreciated in the art, the co-ligands can be the same ordifferent. Suitable ligands fall into two categories: ligands which usenitrogen, oxygen; sulfur, carbon or phosphorus atoms (depending on themetal ion) as the coordination atoms (generally referred to in theliterature as sigma (σ) donors) and organometallic ligands such asmetallocene ligands (generally referred to in the literature as pi (π)donors, and depicted herein as L_(m)). Suitable nitrogen donatingligands are well known in the art and include, but are not limited to,NH₂; NHR; NRR′; pyridine; pyrazine; isonicotinamide; imidazole;bipyridine and substituted derivatives of bipyridine; terpyridine andsubstituted derivatives; phenanthrolines, particularly1,10-phenanthroline (abbreviated phen) and substituted derivatives ofphenanthrolines such as 4,7-dimethylphenanthroline anddipyridol[3,2-a:2′,3′-c]phenazine (abbreviated dppz); dipyridophenazine;1,4,5,8,9,12-hexaazatriphenylene (abbreviated hat);9,10-phenanthrenequinone diimine (abbreviated phi);1,4,5,8-tetraazaphenanthrene (abbreviated tap);1,4,8,11-tetra-azacyclotetradecane (abbreviated cyclam), EDTA, EGTA andisocyanide. Substituted derivatives, including fused derivatives, mayalso be used. In some embodiments, porphyrins and substitutedderivatives of the porphyrin family may be used. See for example,Comprehensive Coordination Chemistry, Ed. Wilkinson et al., PergammonPress, 1987, Chapters 13.2 (pp 73-98), 21.1 (pp. 813-898) and 21.3 (pp915-957), all of which are hereby expressly incorporated by reference.

Suitable sigma donating ligands using carbon, oxygen, sulfur andphosphorus are known in the art. For example, suitable sigma carbondonors are found in Cotton and Wilkenson, Advanced Organic Chemistry,5th Edition, John Wiley & Sons, 1988, hereby incorporated by reference;see page 38, for example. Similarly, suitable oxygen ligands includecrown ethers, water and others known in the art. Phosphines andsubstituted phosphines are also suitable; see page 38 of Cotton andWilkenson.

The oxygen, sulfur, phosphorus and nitrogen-donating ligands areattached in such a manner as to allow the heteroatoms to serve ascoordination atoms.

In a preferred embodiment, organometallic ligands are used. In additionto purely organic compounds for use as redox moieties, and varioustransition metal coordination complexes with δ-bonded organic ligandwith donor atoms as heterocyclic or exocyclic substituents, there isavailable a wide variety of transition metal organometallic compoundswith n-bonded organic ligands (see Advanced Inorganic Chemistry, 5thEd., Cotton & Wilkinson, John Wiley & Sons, 1988, chapter 26;Organometallics, A Concise Introduction, Elschenbroich et al., 2nd Ed.,1992, VCH; and Comprehensive Organometallic Chemistry II, A Review ofthe Literature 1982-1994, Abel et al. Ed., Vol. 7, chapters 7, 8, 10 &11, Pergamon Press, hereby expressly incorporated by reference). Suchorganometallic ligands include cyclic aromatic compounds such as thecyclopentadienide ion [C₅H₅(−1)] and various ring substituted and ringfused derivatives, such as the indenylide (−1) ion, that yield a classof bis(cyclopentadieyl) metal compounds, (i.e. the metallocenes); seefor example Robins et al., J. Am. Chem. Soc. 104:1882-1893 (1982); andGassman et al., J. Am. Chem. Soc. 108:4228-4229 (1986), incorporated byreference. Of these, ferrocene [(C₅H₅)₂Fe] and its derivatives areprototypical examples which have been used in a wide variety of chemical(Connelly et al., Chem. Rev. 96:877-910 (1996), incorporated byreference) and electrochemical (Geiger et al., Advances inOrganometallic Chemistry 23:1-93; and Geiger et al., Advances inOrganometallic Chemistry 24:87, incorporated by reference) electrontransfer or “redox” reactions. Metallocene derivatives of a variety ofthe first, second and third row transition metals are potentialcandidates as redox moieties that are covalently attached to either theribose ring or the nucleoside base of nucleic acid. Other potentiallysuitable organometallic ligands include cyclic arenes such as benzene,to yield bis(arene)metal compounds and their ring substituted and ringfused derivatives, of which bis(benzene)chromium is a prototypicalexample, Other acyclic π-bonded ligands such as the allyl(−1) ion, orbutadiene yield potentially suitable organometallic compounds, and allsuch ligands, in conjuction with other π-bonded and δ-bonded ligandsconstitute the general class of organometallic compounds in which thereis a metal to carbon bond. Electrochemical studies of various dimers andoligomers of such compounds with bridging organic ligands, andadditional non-bridging ligands, as well as with and without metal-metalbonds are potential candidate redox moieties in nucleic acid analysis.

When one or more of the co-ligands is an organometallic ligand, theligand is generally attached via one of the carbon atoms of theorganometallic ligand, although attachment may be via other atoms forheterocyclic ligands. Preferred organometallic ligands includemetallocene ligands, including substituted derivatives and themetalloceneophanes (see page 1174 of Cotton and Wilkenson, supra). Forexample, derivatives of metallocene ligands such asmethylcyclopentadienyl, with multiple methyl groups being preferred,such as pentamethylcyclopentadienyl, can be used to increase thestability of the metallocene. In a preferred embodiment, only one of thetwo metallocene ligands of a metallocene are derivatized.

As described herein, any combination of ligands may be used. Preferredcombinations include: a) all ligands are nitrogen donating ligands; b)all ligands are organometallic ligands; and c) the ligand at theterminus of the conductive oligomer is a metallocene ligand and theligand provided by the nucleic acid is a nitrogen donating ligand, withthe other ligands, if needed, are either nitrogen donating ligands ormetallocene ligands, or a mixture. These combinations are depicted inrepresentative structures using the conductive oligomer of Structure 3are depicted in Structures 27 (using phenanthroline and amino asrepresentative ligands), 28 (using ferrocene as the metal-ligandcombination) and 29 (using cyclopentadienyl and amino as representativeligands).

In addition to serving as attachments for conductive oligomers andelectrodes, the above compositions can also be used as ETM labels. Thatis, as is outlined in FIGS. 19 and 20, transition metals (or other ETMs)attached to conductive oligomers can be added to the nucleic acids fordetection. In this embodiment, without being bound by theory, theconductive oligomer, terminating preferably in an F1 linkage (a linkagethat allows the attachment of the conductive oligomer to the surface),will penetrate the SAM and facilitate electron transfer between the ETMand the electrode. Without being bound by theory, this appears to allowrapid electron transfer, similar to a “mechanism-1” system, by providinga direct pathway for electrons; this is sometimes referred to herein as“hardwiring”.

Surprisingly, the system appears to work whether or not the F1 moiety isprotected; that is, a direct attachment may not be required to increasethe frequency response of the ETM. Thus, the conductive oligomer canterminate either in an F1 moiety, an F1 moiety protected with aprotecting group (see Greene, supra), or need not terminate in an F1moiety at all; terminal groups such as are used on the surfaces of theSAMs may also be used. Alternatively, the bare terminus of theconductive oligomer may be sufficient.

In this embodiment, a plurality of ETMs per “branch” may be used. Theymay be attached as a group, e.g. as a metallocene polymer, terminatingin the conductive oligomer, or may be substitution groups off of theconductive oligomer. In general, preferred embodiments utilizeelectronic conjugation between the ETMs and the conductive oligomer, tofacilitate electron transfer.

In general, the length of the conductive oligomer in this embodimentwill vary with the length of the SAM on the electrode, and preferredembodiments utilize two unit and three unit oligomers. Preferredconductive oligomers in this embodiment are the same as those outlinedabove for attachment of nucleic acids to electrodes, withphenyl-acetylene oligomers being the most preferred.

In this embodiment, the ETM with the attached conductive oligomer isgenerally synthesized, and then a phosphoramidite moiety is made.

In a preferred embodiment, the ligands used in the invention showaltered fluoroscent properties depending on the redox state of thechelated metal ion. As described below, this thus serves as anadditional mode of detection of electron transfer between the ETM andthe electrode.

In a preferred embodiment, as is described more fully below, the ligandattached to the nucleic acid is an amino group attached to the 2′ or 3′position of a ribose of the ribose-phosphate backbone. This ligand maycontain a multiplicity of amino groups so as to form a polydentateligand which binds the metal ion. Other preferred ligands includecyclopentadiene and phenanthroline.

The use of metal ions to connect the nucleic acids can serve as aninternal control or calibration of the system, to evaluate the number ofavailable nucleic acids on the surface. However, as will be appreciatedby those in the art, if metal ions are used to connect the nucleic acidsto the conductive oligomers, it is generally desirable to have thismetal ion complex have a different redox potential than that of the ETMsused in the rest of the system, as described below. This is generallytrue so as to be able to distinguish the presence of the capture probefrom the presence of the target sequence. This may be useful foridentification, calibration and/or quantification. Thus, the amount ofcapture probe on an electrode may be compared to the amount ofhybridized double stranded nucleic acid to quantify the amount of targetsequence in a sample. This is quite significant to serve as an internalcontrol of the sensor or system. This allows a measurement either priorto the addition of target or after, on the same molecules that will beused for detection, rather than rely on a similar but different controlsystem. Thus, the actual molecules that will be used for the detectioncan be quantified prior to any experiment. This is a significantadvantage over prior methods.

In a preferred embodiment, the capture probe nucleic acids arecovalently attached to the electrode via an insulator. The attachment ofnucleic acids to insulators such as alkyl groups is well known, and canbe done to the base or the backbone, including the ribose or phosphatefor backbones containing these moieties, or to alternate backbones fornucleic acid analogs.

In a preferred embodiment, there may be one or more different captureprobe species on the surface. In some embodiments, there may be one typeof capture probe, or one type of capture probe extender, as is morefully described below. Alternatively, different capture probes, or onecapture probes with a multiplicity of different capture extender probescan be used. Similarly, it may be desirable to use auxiliary captureprobes that comprise relatively short probe sequences, that can be usedto “tack down” components of the system, for example the recruitmentlinkers, to increase the concentration of ETMs at the surface.

Thus the present invention provides electrodes, preferably comprisingmonolayers and capture binding ligands, useful in target analytedetection systems.

In a preferred embodiment, the assay complex comprises a first transportcomponent comprising a first binding partner. The first transportcomponent is used to transport the assay complex comprising the firstcomponent to the electrode, as is more described herein.

In a preferred embodiment, the first component is a particle. By“particle” or “microparticle” or “nanoparticle” or “bead” or“microsphere” herein is meant microparticulate matter. As will beappreciated by those in the art, the first particles can comprise a widevariety of materials, including, but not limited to, cross-linkedstarch, dextrans, cellulose, proteins, organic polymers includingstyrene polymers including polystyrene and methylstyrene as well asother styrene co-polymers, plastics, glass, ceramics, acrylic polymers,magnetically responsive materials, colloids, thoria sol, carbongraphite, titanium dioxide, nylon, latex, and teflon may all be used.“Microsphere Detection Guide” from Bangs Laboratories, Fishers Ind. is ahelpful guide. Preferred embodiments utilize magnetic particles andcolloids.

The size of the particles will depend on their composition. Theparticles need not be spherical; irregular particles may be used. Inaddition, the particles may be porous, thus increasing the surface areaof the particle available for attachment of binding ligands, capturemoieties, or ETMs. In general, the size of the particles will vary withtheir composition; for example, magnetic particles are generally biggerthan colloid particles. Thus, the particles have diameters ranging from1-5 nm (colloids) to 200 μm (magnetic particles).

In a preferred embodiment, the first particle is a magnetic particle ora particle that can be induced to display magnetic properties. By“magnetic” herein is meant that the particle is attracted in a magneticfield, including ferromagnetic, paramagnetic, and diamagnetic. In thisembodiment, the particles are preferably from about 0.001 to about 200μm in diameter, with from about 0.05 to about 200 μm preferred, fromabout 0.1 to about 100 μm being particularly preferred, and from about0.5 to about 10 μm being especially preferred.

In a preferred embodiment, the first particle is a colloid particle ornanoparticle. By “colloid” or “nanoparticle” herein is meant a particlethat is small enough to stay suspended in solution in standard solventsand standard conditions. Generally, a colloid is a particle in the sizerange of 1 nm to 1 μm. A suspension of colloids is distinguishable fromnon-colloids in that their spatial distribution is largely unaltered bygravity during the time course of an experimental observation. As isknown in the art, colloids can be made from a variety of materials; seeSchmid, ed. Clusters and Colloids, VCH, Weinheim, 1994; Hayat ed.Colloidal Gold: Principles, Methods and Applications (Academic, SanDiego, 1991); Bassell et al., J. Cell Biol. 126:863 1994; and Creightonet al., J. Chem. Soc. Faraday II 75:790 (1979), all of which are herebyincorporated by reference. Preferred colloids include, but are notlimited to, those of Au, Se, Te, Co, Ni, Fe, Cu, Pt and other transitionmetals, and other colloids known in the art. It is known that manycolloid particles are charged and thus naturally repel each other; forexample, Au colloid particles are generally negatively charged. Thisfacilitates non-aggregation except in the presence of target analyte asdescribed below.

In some embodiments, the transport composition is not a particle, and isin solution. In this embodiment, the transport composition comprises acapture ligand as outlined below.

The first components comprise a first binding partner. By “bindingpartner” or “binding ligand” or grammatical equivalents herein is meanta compound that is used to probe for the presence of the target analyte,and that will bind to the target analyte. As for the capture bindingligands outlined herein, the composition of the binding ligand willdepend on the composition of the target analyte, as generally describedabove.

Generally, the first binding ligands are attached to the transportcomponent in a variety of ways, depending on whether the transportcomponent is a particle, the composition of the particle, and thecomposition of the binding ligand.

In a preferred embodiment, the transport component is a particle, andattachment proceeds on the basis of the composition of the particle andthe composition of the first binding ligand. In a preferred embodiment,depending on the composition of the particle, it will contain chemicalfunctional groups for subsequent attachment of other moieties. Forexample, when the particle is a magnetic particle, magnetic particleswith chemical functional groups such as amines are commerciallyavailable. Preferred functional groups for attachment are amino groups,carboxy groups, oxo groups and thiol groups, with amino groups beingparticularly preferred. Using these functional groups, the bindingligands can be attached using functional groups on the binding ligands.For example, proteins and nucleic acids containing amino groups can beattached to particles comprising amino groups, for example using linkersas are known in the art; for example, homo- or hetero-bifunctionallinkers as are well known (see 1994 Pierce Chemical Company catalog,technical section on cross-linkers, pages 155-200, incorporated hereinby reference).

When the transport particle is a colloid particle, attachment will againdepend on the composition of the colloid and of the binding ligand, andgenerally proceeds as outlined above for attachment to electrodes.

The density of the binding ligand on the particle can vary widely, andwill depend in part on the presence or absence of the other componentson the particle, including, but not limited to, capture binding ligands,SAMs including conductive oligomers and insulators, charged moieties,etc. In general, when large target analytes are to be detected, such aslarge proteins or long nucleic acids, the density of the binding ligandis decreased, to allow sufficient “space” for the target analyte tobind.

In one embodiment, the transport component comprises a capture ligandthat can be used for attachment to an electrode that comprises a capturebinding partner. As defined herein for binding ligands, capture ligandsand capture binding partners are pairs that specifically interact, suchthat the transport composition can be attached to the electrode. Captureligands and capture binding partners may comprising a large number ofdifferent species, as is outlined above for binding ligands, withproteins and nucleic acids being particularly preferred. Thus, thecapture ligand and capture binding partner can be used to transport theassay complex to the electrode surface, as is more fully describedbelow.

In a preferred embodiment, the transport component is not a particle,and instead comprises a capture binding ligand and a first bindingligand. Again, the method of attachment will depend on the compositionof each. In general, a variety of methods are known for the attachmentof two proteins, including the use of linkers including polymers, andhomo- and heterobifunctional linkers as described herein. In some cases,recombinant DNA technology can be utilized to make fusion proteinscomprising at least a first domain comprising the first binding ligandand a second domain comprising the capture binding ligand. Similarly,when the binding ligand and capture binding ligand are both, nucleicacids, they may be generally contiguous sequences that are synthesizedas such. Preferably in this embodiment, the reporter composition is aparticle, preferably a gold colloid particle with a SAM including ETMs.

In a preferred embodiment, when the transport component does notcomprise a particle, it is possible to use polymers, either linear orbranched, that have a plurality of first binding ligands attached, usingtechniques known in the art. Upon binding of a plurality of targetmolecules and the reporter compositions, particularly when the reportercompositions comprise particles, it is possible to effect aggregation ofthe assay complex, leading to transport to the electrode surface. Thus,in this embodiment, there may or may not be capture binding ligands.Preferably in this embodiment, the reporter composition is a particle,preferably a gold colloid particle with a SAM including ETMs.

In a preferred embodiment, the transport particle further comprises aself-assembled monolayer (SAM), particularly when the transport particleis a gold colloid particle. By “monolayer” or “self-assembled monolayer”or “SAM” herein is meant a relatively ordered assembly of moleculesspontaneously chemisorbed on a surface, in which the molecules areoriented approximately parallel to each other and roughly perpendicularto the surface. Each of the molecules includes a functional group thatadheres to the surface, and a portion that interacts with neighboringmolecules in the monolayer to form the relatively ordered array. A“mixed” monolayer comprises a heterogeneous monolayer, that is, where atleast two different molecules make up the monolayer. The SAM maycomprise attachment linkers, including conductive oligomers andinsulators either together or each separately, and the composition ofthe SAM may depend on its location; that is, as is more fully describedbelow, SAMs on the electrode must comprise at least some conductiveoligomers. As outlined herein, the efficiency of oligonucleotidehybridization, and other binding events, may increase when the analyteis at a distance from the surface. Similarly, non-specific binding ofbiomolecules, including nucleic acids, to a surface is generally reducedwhen a monolayer is present. Thus, a monolayer facilitates themaintenance of the analyte away from the surface. In addition, when theSAM is on the electrode, a monolayer serves to keep charge carriers awayfrom the surface of the electrode. Thus, this layer helps to preventdirect electrical contact between the electrode and charged specieswithin the solvent. Such contact can result in a direct “short circuit”or an indirect short circuit via charged species which may be present inthe sample. Accordingly, the monolayer is preferably tightly packed in auniform layer on the electrode surface, such that a minimum of “holes”exist. The monolayer thus serves as a physical barrier to block solventaccesibility to the electrode.

As will be appreciated by those in the art, the actual combinations andratios of the different species making up the monolayer can vary widely,and will depend on where the monolayer is, i.e. on a transport particle,a reporter particle, or on the electrode. For attachment on a transportparticle, generally two, three or four component systems are preferred.The components are as follows. The first species comprises a bindingligand containing species (that is generally attached to the surface viaan attachment linker, i.e. either an insulator or a conductive oligomer;as is more fully described below). The second species are the conductiveoligomers. The third species are insulators. The fourth species is acharged species that will prevent the particles of the invention fromaggregating in the absence of the target analyte; this may or may not berequired, depending on the particle and the binding ligand; that is, insome instances the binding ligand may comprise a charged species. Forexample, when the binding ligand is a nucleic acid, the nucleic acidsprovide the required charge and a fourth species is not required. Thesecond, third and fourth species are optional on a transport particle,particularly a magnetic particle.

A representative three component system comprises the first, second andthird species. In this embodiment, the actual ratio of the componentswill vary, in part depending on the size of the binding ligand andtarget analyte; i.e. for larger ligands or analytes, a lower amount offirst species is desirable. Thus, a preferred embodiment utilizes athree component system with the first species comprising from about 90%to about 1%, with from about 20% to about 40% being preferred, and fromabout 30% to about 40% being especially preferred for small targets andfrom about 10% to about 20% preferred for larger targets. The secondspecies can comprise from about 1% to about 90%, with from about 20% toabout 90% being preferred, and from about 40% to about 60% beingespecially preferred. The third species can comprise from about 1% toabout 90%, with from about 20% to about 40% being preferred, and fromabout 15% to about 30% being especially preferred. Preferred ratios offirst:second:third species are 2:2:1 for small targets, 1:3:1 for largertargets, with total thiol concentration in the 500 μM to 1 mM range, and833 μM being preferred.

In a preferred embodiment, two component systems are used, comprisingthe first and second species. In this embodiment, the first species cancomprise from about 90% to about 1%, with from about 1% to about 40%being preferred, and from about 10% to about 40% being especiallypreferred. The second species can comprise from about 1% to about 90%,with from about 10% to about 60% being preferred, and from about 20% toabout 40% being especially preferred.

In some embodiments, it is possible to avoid the use of a firsttransport component completely. What is important in this embodiment isthat the reporter compositions are unable to associate with theelectrode in the absence of target analyte. This can be done in twodifferent ways. In one embodiment, two reporter particles are used; thatis, when the aggregation of the particles is used to transport the assaycomplex to the electrode as is more fully outlined below, it is possibleto use a first and a second reporter particle, each containing a bindingligand for a different portion of the target analyte and each containingETMs, as is generally depicted in FIG. 1F. In this embodiment, thespecificity of the system is achieved through the use of a reporterparticles that will not settle in the absence of aggregation. That is,in the absence of target analyte, the two reporter particles staysuspended in solution; upon the introduction of the target analyte,large “cross-linked” aggregation assay complexes are formed that cansettle or be brought into contact with the electrode. Alternatively, itis possible to use the target analyte as the transport component; thatis, part of the target analyte is used as a capture ligand, as describedabove and depicted in FIGS. 1E and 2, with one or more reportercompositions.

Generally, the first transport component does not comprise any ETMlabels to decrease the amount of background signal. However, it ispossible to use transport components that do comprise ETMs; in thisembodiment, it is important that the first transport component does notcontain the same ETM labels that are used in the reporter components.For example, ETM labels with different redox potentials may be used,such that the ETM labels on the reporter components are distinguishablefrom the labels on the transport components. Alternatively, other typesof labels, such as fluorescent labels, may be used on the components.This may find use in the development of controls and calibration of thesystem.

In addition to the first components, the compositions of the inventionfurther comprise a reporter composition that is used to signal thepresence of the target analyte.

In a preferred embodiment, the reporter composition comprises a secondparticle. As defined above, the second particle may comprise a largenumber of different compositions. However, if the transport mechanismused is magnetic attraction, it is preferred in this embodiment that thesecond particle is non-magnetic.

In a preferred embodiment, the second particle is a colloid particle asdefined above. Particularly preferred are gold colloid particles, due tothe ease of attachment of sulfur-containing moieties.

In a preferred embodiment, the reporter composition is not a particle.In this embodiment, the reporter composition comprises a second bindingligand and at least one ETM, as defined below and shown in FIGS. 3, 4and 5. Generally in this embodiment the reporter composition comprises arecruitment linker that has attached ETMs.

The reporter composition comprises a second binding ligand, as outlinedabove. The second binding ligand can be the same or different from thefirst binding ligand. In addition, the compositions of the invention(including both the transport and the reporter compositions), maycomprise multiple different binding ligands.

The reporter composition further comprises at least one, and preferablya plurality, of signalling moieties or labels that can be used to detectthe reporter compositions or assay complexes containing them,particularly reporter particles including colloids. Suitable signallingmoieties include any detectable labels, including, but not limited to,labels detectable optically, fluorescently, electronically,electrochemically, radioactively, labels detectable viachemiluminescence, electrochemiluminesce, enzymes,fluorescence-resonance energy transfer (FRET), and RAMAN techniques.Thus, signalling moieties include, but are not limited to, electrontransfer moieties, fluorescent moieties, radioisotopic moieties, opticaldyes, RAMAN labels, etc.

In a preferred embodiment, the signalling moieties are electron transfermoieties. The terms “electron donor moiety”, “electron acceptor moiety”,and “electron transfer moieties” (ETMs) or grammatical equivalentsherein refers to molecules capable of electron transfer under certainconditions. It is to be understood that electron donor and acceptorcapabilities are relative; that is, a molecule which can lose anelectron under certain experimental conditions will be able to accept anelectron under different experimental conditions. It is to be understoodthat the number of possible electron donor moieties and electronacceptor moieties is very large, and that one skilled in the art ofelectron transfer compounds will be able to utilize a number ofcompounds in the present invention. Preferred ETMs include, but are notlimited to, transition metal complexes, organic ETMs, and electrodes.

In a preferred embodiment, the ETMs are transition metal complexes.Transition metals are those whose atoms have a partial or complete dshell of electrons. Suitable transition metals for use in the inventioninclude, but are not limited to, cadmium (Cd), copper (Cu), cobalt (Co),palladium (Pd), zinc (Zn), iron (Fe), ruthenium (Ru), rhodium (Rh),osmium (Os), rhenium (Re), platinium (Pt), scandium (Sc), titanium (Ti),Vanadium (V), chromium (Cr), manganese (Mn), nickel (Ni), Molybdenum(Mo), technetium (Tc), tungsten (W), and iridium (Ir). That is, thefirst series of transition metals, the platinum metals (Ru, Rh, Pd, Os,Ir and Pt), along with Fe, Re, W, Mo and Tc, are preferred. Particularlypreferred are ruthenium, rhenium, osmium, platinium, cobalt and iron.

The transition metals are complexed with a variety of ligands, L, toform suitable transition metal complexes, as is well known in the art.The ligands provide the coordination atoms for the binding of the metalion. As will be appreciated in the art, the co-ligands can be the sameor different. Suitable ligands fall into two categories: ligands whichuse nitrogen, oxygen, sulfur, carbon or phosphorus atoms (depending onthe metal ion) as the coordination atoms (generally referred to in theliterature as sigma (a) donors) and organometallic ligands such asmetallocene ligands (generally referred to in the literature as pi (n)donors, and depicted herein as L_(m)). Suitable nitrogen donatingligands are well known in the art and include, but are not limited to,NH₂; NHR; NRR′; pyridine; pyrazine; isonicotinamide; imidazole;bipyridine and substituted derivatives of bipyridine; terpyridine andsubstituted derivatives; phenanthrolines, particularly1,10-phenanthroline (abbreviated phen) and substituted derivatives ofphenanthrolines such as 4,7-dimethylphenanthroline anddipyridol[3,2-a:2′,3′-c]phenazine (abbreviated dppz); dipyridophenazine;1,4,5,8,9,12-hexaazatriphenylene (abbreviated hat);9,10-phenanthrenequinone diimine (abbreviated phi);1,4,5,8-tetraazaphenanthrene (abbreviated tap);1,4,8,11-tetra-azacyclotetradecane (abbreviated cyclam), EDTA, EGTA andisocyanide. Substituted derivatives, including fused derivatives, mayalso be used. In some embodiments, porphyrins and substitutedderivatives of the porphyrin family may be used. See for example,Comprehensive Coordination Chemistry, Ed. Wilkinson et al., PergammonPress, 1987, Chapters 13.2 (pp 73-98), 21.1 (pp. 813-898) and 21.3 (pp915-957), all of which are hereby expressly incorporated by reference.

Suitable sigma donating ligands using carbon, oxygen, sulfur andphosphorus are known in the art. For example, suitable sigma carbondonors are found in Cotton and Wilkenson, Advanced Organic Chemistry,5th Edition, John Wiley & Sons, 1988, hereby incorporated by reference;see page 38, for example. Similarly, suitable oxygen ligands includecrown ethers, water and others known in the art. Phosphines andsubstituted phosphines are also suitable; see page 38 of Cotton andWilkenson.

The oxygen, sulfur, phosphorus and nitrogen-donating ligands areattached in such a manner as to allow the heteroatoms to serve ascoordination atoms.

In a preferred embodiment, organometallic ligands are used. In additionto purely organic compounds for use as redox moieties, and varioustransition metal coordination complexes with δ-bonded organic ligandwith donor atoms as heterocyclic or exocyclic substituents, there isavailable a wide variety of transition metal organometallic compoundswith n-bonded organic ligands (see Advanced Inorganic Chemistry, 5thEd., Cotton & Wilkinson, John Wiley & Sons, 1988, chapter 26;Organometallics, A Concise Introduction, Elschenbroich et al., 2nd Ed.,1992, VCH; and Comprehensive Organometallic Chemistry II, A Review ofthe Literature 1982-1994, Abel et al. Ed., Vol. 7, chapters 7, 8, 10 &11, Pergamon Press, hereby expressly incorporated by reference). Suchorganometallic ligands include cyclic aromatic compounds such as thecyclopentadienide ion [C₅H₅(−1)] and various ring substituted and ringfused derivatives, such as the indenylide (−1) ion, that yield a classof bis(cyclopentadieyl) metal compounds, (i.e. the metallocenes); seefor example Robins et al., J. Am. Chem. Soc. 104:1882-1893 (1982); andGassman et al., J. Am. Chem. Soc. 108:4228-4229 (1986), incorporated byreference. Of these, ferrocene [(C₅H₅)₂Fe] and its derivatives areprototypical examples which have been used in a wide variety of chemical(Connelly et al., Chem. Rev. 96:877-910 (1996), incorporated byreference) and electrochemical (Geiger et al., Advances inOrganometallic Chemistry 23:1-93; and Geiger et al., Advances inOrganometallic Chemistry 24:87, incorporated by reference) electrontransfer or “redox” reactions: Metallocene derivatives of a variety ofthe first, second and third row transition metals are potentialcandidates as redox moieties that are covalently attached to either theribose ring or the nucleoside base of nucleic acid. Other potentiallysuitable organometallic ligands include cyclic arenes such as benzene,to yield bis(arene)metal compounds and their ring substituted and ringfused derivatives, of which bis(benzene)chromium is a prototypicalexample, Other acyclic n-bonded ligands such as the allyl(−1) ion, orbutadiene yield potentially suitable organometallic compounds, and allsuch ligands, in conjuction with other n-bonded and δ-bonded ligandsconstitute the general class of organometallic compounds in which thereis a metal to carbon bond. Electrochemical studies of various dimers andoligomers of such compounds with bridging organic ligands, andadditional non-bridging ligands, as well as with and without metal-metalbonds are potential candidate redox moieties in nucleic acid analysis.

When one or more of the co-ligands is an organometallic ligand, theligand is generally attached via one of the carbon atoms of theorganometallic ligand, although attachment may be via other atoms forheterocyclic ligands. Preferred organometallic ligands includemetallocene ligands, including substituted derivatives and themetalloceneophanes (see page 1174 of Cotton and Wilkenson, supra). Forexample, derivatives of metallocene ligands such asmethylcyclopentadienyl, with multiple methyl groups being preferred,such as pentamethylcyclopentadienyl, can be used to increase thestability of the metallocene. In a preferred embodiment, only one of thetwo metallocene ligands of a metallocene are derivatized.

As described herein, any combination of ligands may be used. Preferredcombinations include: a) all ligands are nitrogen donating ligands; b)all ligands are organometallic ligands; and c) the ligand at theterminus of the conductive oligomer is a metallocene ligand and theligand provided by the nucleic acid is a nitrogen donating ligand, withthe other ligands, if needed, are either nitrogen donating ligands ormetallocene ligands, or a mixture.

In addition to transition metal complexes, other organic electron donorsand acceptors may be attached for use in the invention. These organicmolecules include, but are not limited to, riboflavin, xanthene dyes,azine dyes, acridine orange, N,N′-dimethyl-2,7-diazapyrenium dichloride(DAP²⁺), methylviologen, ethidium bromide, quinones such asN,N-dimethylanthra(2,1,9-def:6,5,10-d′e′f′)diisoquinoline dichloride(ADIQ²⁺); porphyrins ([meso-tetrakis(N-methyl-x-pyridinium)porphyrintetrachloride], varlamine blue B hydrochloride, Bindschedler's green;2,6-dichloroindophenol, 2,6-dibromophenolindophenol; Brilliant crestblue (3-amino-9-dimethyl-amino-10-methylphenoxyazine chloride),methylene blue; Nile blue A (aminoaphthodiethylaminophenoxazinesulfate), indigo-5,5′,7,7′-tetrasulfonic acid, indigo-5,5′,7-trisulfonicacid; phenosafranine, indigo-5-monosulfonic acid; safranine T;bis(dimethylglyoximato)-iron(II) chloride; induline scarlet, neutralred, anthracene, coronene, pyrene, 9-phenylanthracene, rubrene,binaphthyl, DPA, phenothiazene, fluoranthene, phenanthrene, chrysene,1,8-diphenyl-1,3,5,7-octatetracene, naphthalene, acenaphthalene,perylene, TMPD and analogs and subsitituted derivatives of thesecompounds.

In one embodiment, the electron donors and acceptors are redox proteinsas are known in the art. However, redox proteins in many embodiments arenot preferred.

The number of ETMs per reporter composition can vary, and can depend onthe composition of the reporter composition (i.e. whether it is aparticle) and the method of attachment, as is more fully describedbelow. In a preferred embodiment, a plurality of ETMs are used. As isshown in the examples, the use of multiple ETMs provides signalamplification and thus allows more sensitive detection limits. Generallyfrom one to millions or more can be used.

The ETMs may be attached to the reporter compositions in a variety ofways, including methods described in WO 98/20162 and U.S. Ser. No.09/135,183, filed Aug. 17, 1998, both of which are hereby incorporatedby reference in their entirety. In a preferred embodiment, as is morefully outlined below, it is desirable to attach the ETMs to the reportercomposition in a manner that may maximize “cross-talk” as betweendifferent ETMs; that is, to maximize the possible conjugation of thesystem. This generally involves using either closely packed ETMs, as isgenerally depicted in FIGS. 4 and 5, or, when using colloid particles,conductive oligomers to attach the ETMs to the colloid particle, as isgenerally depicted in FIG. 1. Without being bound by theory, it appearsthat the use of ETMs attached to the metallic colloid particle viaconductive oligomers allows electron transfer between ETMs on the entireparticle rather than just those in spatial proximity to the electrode;that is the system is conjugated, allowing “access” of most or all ofthe ETMs on a particular reporter composition.

Accordingly, in a preferred embodiment, the ETM is attached to thereporter composition in a variety of ways.

In a preferred embodiment, the ETM is attached directly to the reportercomposition, i.e. directly to the particle. This may be done as isgenerally outlined above for the attachment of binding ligands tomagnetic particles or other particles, using functional groups on boththe particle and the ETMs for attachment.

In a preferred embodiment, the reporter composition is a particle, andthe ETM is attached via an attachment linker. The method of attachmentwill depend on the composition of the ETM and the attachment linker aswill be appreciated by those in the art. In a preferred embodiment, thereporter composition is a particle and the attachment linker is aconductive oligomer. Preferred methods of attachment for this embodimentwhen the binding ligand is a nucleic acid are outlined in WO 98/20162,hereby incorporated by reference in its entirety; see particularlystructures 31-34.

In a preferred embodiment, the ETM is attached to the terminus of theattachment linker, as depicted below in Structures 17, 18, 19 and 20.These structures depict the preferred embodiment of conductive oligomersas the attachment linkers, although insulators are useful as well. Inaddition, these structures depict metallocenes as the ETMs, although aswill be appreciated by those in the art, other ETMs may be used as well.These structures depict the use of a single ETM per attachment linker,although as will be appreciated by those in the art, “branched”attachment linkers may also be used to result in multiple ETMs at thetermini of the linker; in addition, ETMs may be attached as substitutiongroups along the length of the linker as well. In this embodiment, theattachment linkers basically form a SAM.

Structure 17 utilizes a Structure 10 conductive oligomer, although aswill be appreciated by those in the art, other conductive oligomers maybe used. Preferred embodiments of Structure 17 are depicted below.

Preferred R groups of Structure 19 are hydrogen.

These compositions are synthesized as follows. The conductive oligomerlinked to the metallocene is made as described herein; see also, Hsunget al., Organometallics 14:4808-4815 (1995); and Bumm et al., Science271:1705 (1996), both of which are expressly incorporated herein byreference. The conductive oligomer is then attached to the electrodeusing the novel ethylpyridine protecting group, as outlined herein.

Alternatively, the ETMs can be attached as a plurality of ETMs as isgenerally outlined below for non-particle reporter compositions, usingcomponents as is generally outlined in FIG. 3.

In a preferred embodiment, the reporter composition is a particle andthe attachment linker is an insulator. This is generally done using theabove techniques as will be appreciated by those in the art. In apreferred embodiment, the reporter composition is not a particle and thebinding ligand is linked either directly or indirectly to at least oneETM. This can be done directly, by attaching the binding ligand directlyto the ETM, again generally by using chemical functionalities on each,as is generally described above. Similarly, linkers can be used,including polymers and homo- and heterobifunctional tinkers as describedherein.

In a preferred embodiment, a recruitment linker comprising a pluralityof ETMs is used, either for attachment to the binding ligand or forattachment to a particle comprising the binding ligand. This isparticularly preferred when a particle is not used to allow more signalper target analyte, although as outlined above, these techniques can beused with particles as well. In this embodiment, the recruitment linkercan be virtually any polymer, with nucleic acid being preferred,particularly when a particle is not used and the ETMs are to be linkeddirectly to a nucleic acid binding ligand; this allows the synthesis ofthe reporter composition to be done in one step. In some embodiments, asis more fully outlined below, the recruitment linker may comprisedouble-stranded portions.

Thus, as will be appreciated by those in the art, there are a variety ofconfigurations that can be used. In a preferred embodiment, therecruitment linker is nucleic acid (including analogs), and attachmentof the ETMs can be via (1) a base; (2) the backbone, including theribose, the phosphate, or comparable structures in nucleic acid analogs;(3) nucleoside replacement, described below; or (4) metallocenepolymers, as described below. In a preferred embodiment, the recruitmentlinker is non-nucleic acid, and can be either a metallocene polymer oran alkyl-type polymer (including heteroalkyl, as is more fully describedbelow) containing ETM substitution groups. These options are generallydepicted in FIG. 3.

In a preferred embodiment, the recruitment linker is a nucleic acid, andcomprises covalently attached ETMs. The ETMs may be attached tonucleosides within the nucleic acid in a variety of positions. Preferredembodiments include, but are not limited to, (1) attachment to the baseof the nucleoside, (2) attachment of the ETM as a base replacement, (3)attachment to the backbone of the nucleic acid, including either to aribose of the ribose-phosphate backbone or to a phosphate moiety, or toanalogous structures in nucleic acid analogs, and (4) attachment viametallocene polymers, with the latter being preferred.

In a preferred embodiment, the ETM is attached to the base of anucleoside as is generally outlined herein and in WO 98/20162 for theattachment of conductive oligomers. Attachment can be to an internalnucleoside or a terminal nucleoside, or combinations of these.

Ligands containing aromatic groups can be attached via acetylenelinkages as is known in the art (see Comprehensive Organic Synthesis,Trost et al., Ed., Pergamon Press, Chapter 2.4: Coupling ReactionsBetween sp² and sp Carbon Centers, Sonogashira, pp 521-549, and pp950-953, hereby incorporated by reference). Structure 21 depicts arepresentative structure in the presence of the metal ion and any othernecessary ligands; Structure 21 depicts uridine, although as for all thestructures herein, any other base may also be used.

L_(a) is a ligand, which may include nitrogen, oxygen, sulfur orphosphorus donating ligands or organometallic ligands such asmetallocene ligands. Suitable L_(a) ligands include, but not limited to,phenanthroline, imidazole, bpy and terpy. L, and M are as defined above.Again, it will be appreciated by those in the art, a linker (“Z”) may beincluded between the nucleoside and the ETM.

Similarly, as for the conductive oligomers, the linkage may be doneusing a linker, which may utilize an amide linkage (see generally Telseret al., J. Am. Chem. Soc. 111:7221-7226 (1989); Telser et al., J. Am.Chem. Soc. 111:7226-7232 (1989), both of which are expresslyincorporated by reference). These structures are generally depictedbelow in Structure 22, which again uses uridine as the base, although asabove, the other bases may also be used:

In this embodiment, L is a ligand as defined above, with L_(r) and M asdefined above as well. Preferably, L is amino, phen, byp and terpy.

In a preferred embodiment, the ETM attached to a nucleoside is ametallocene; i.e. the L and L, of Structure 22 are both metalloceneligands, L_(m), as described above. Structure 23 depicts a preferredembodiment wherein the metallocene is ferrocene, and the base isuridine, although other bases may be used:

Preliminary data suggest that Structure 23 may cyclize, with the secondacetylene carbon atom attacking the carbonyl oxygen, forming afuran-like structure. Preferred metallocenes include ferrocene,cobaltocene and osmiumocene.

In a preferred embodiment, the ETM is attached to a ribose at anyposition of the ribose-phosphate backbone of the nucleic acid, i.e.either the 5′ or 3′ terminus or any internal nucleoside. Ribose in thiscase can include ribose analogs. As is known in the art, nucleosidesthat are modified at either the 2′ or 3′ position of the ribose can bemade, with nitrogen, oxygen, sulfur and phosphorus-containingmodifications possible. Amino-modified and oxygen-modified ribose ispreferred. See generally PCT publication WO 95/15971, incorporatedherein by reference. These modification groups may be used as atransition metal ligand, or as a chemically functional moiety forattachment of other transition metal ligands and organometallic ligands,or organic electron donor moieties as will be appreciated by those inthe art. In this embodiment, a linker such as depicted herein for “Z”may be used as well, or a conductive oligomer between the ribose and theETM. Preferred embodiments utilize attachment at the 2′ or 3′ positionof the ribose, with the 2′ position being preferred. Thus for example,conductive oligomers may be replaced by ETMs; alternatively, the ETMsmay be added to the free terminus of the conductive oligomer.

In a preferred embodiment, a metallocene serves as the ETM, and isattached via an amide bond as depicted below in Structure 24. Theexamples outline the synthesis of a preferred compound when themetallocene is ferrocene.

In a preferred embodiment, amine linkages are used, as is generallydepicted in Structure 25.

Z is a linker, as defined herein, with 1-16 atoms being preferred, and2-4 atoms being particularly preferred, and t is either one or zero.

In a preferred embodiment, oxo linkages are used, as is generallydepicted in Structure 26.

In Structure 26, Z is a linker, as defined herein, and t is either oneor zero. Preferred Z linkers include alkyl groups including heteroalkylgroups such as (CH₂)n and (CH₂CH₂O)n, with n from 1 to 10 beingpreferred, and n=1 to 4 being especially preferred, and n=4 beingparticularly preferred.

Linkages utilizing other heteroatoms are also possible.

In a preferred embodiment, an ETM is attached to a phosphate at anyposition of the ribose-phosphate backbone of the nucleic acid. This maybe done in a variety of ways. In one embodiment, phosphodiester bondanalogs such as phosphoramide or phosphoramidite linkages may beincorporated into a nucleic acid, where the heteroatom (i.e. nitrogen)serves as a transition metal ligand (see PCT publication WO 95/15971,incorporated by reference). Alternatively, the conductive oligomersdepicted in the structures may be replaced by ETMs. In a preferredembodiment, the composition has the structure shown in Structure 25.

In Structure 25, the ETM is attached via a phosphate linkage, generallythrough the use of a linker, Z. Preferred Z linkers include alkylgroups, including heteroalkyl groups such as (CH₂)_(n), (CH₂CH₂O)_(n),with n from 1 to 10 being preferred, and n=1 to 4 being especiallypreferred, and n=4 being particularly preferred.

When the ETM is attached to the base or the backbone of the nucleoside,it is possible to attach the ETMs via “dendrimer” structures, as is morefully outlined below. As is generally depicted in the Figures,alkyl-based linkers can be used to create multiple branching structurescomprising one or more ETMs at the terminus of each branch. Generally,this is done by creating branch points containing multiple hydroxygroups, which optionally can then be used to add additional branchpoints. The terminal hydroxy groups can then be used in phosphoramiditereactions to add ETMs, as is generally done below for the nucleosidereplacement and metallocene polymer reactions.

In a preferred embodiment, an ETM such as a metallocene is used as a“nucleoside replacement”, serving as an ETM. For example, the distancebetween the two cyclopentadiene rings of ferrocene is similar to theorthongonal distance between two bases in a double stranded nucleicacid. Other metallocenes in addition to ferrocene may be used, forexample, air stable metallocenes such as those containing cobalt orruthenium. Thus, metallocene moieties may be incorporated into thebackbone of a nucleic acid, as is generally depicted in Structure 26(nucleic acid with a ribose-phosphate backbone) and Structure 27(peptide nucleic acid backbone). Structures 26 and 27 depict ferrocene,although as will be appreciated by those in the art, other metallocenesmay be used as well. In general, air stable metallocenes are preferred,including metallocenes utilizing ruthenium and cobalt as the metal.

In Structure 26, Z is a linker as defined above, with generally short,alkyl groups, including heteroatoms such as oxygen being preferred.Generally, what is important is the length of the linker, such thatminimal perturbations of a double stranded nucleic acid is effected, asis more fully described below. Thus, methylene, ethylene, ethyleneglycols, propylene and butylene are all preferred, with ethylene andethylene glycol being particularly preferred. In addition, each Z linkermay be the same or different. Structure 26 depicts a ribose-phosphatebackbone, although as will be appreciated by those in the art, nucleicacid analogs may also be used, including ribose analogs and phosphatebond analogs.

In Structure 27, preferred Z groups are as listed above, and again, eachZ linker can be the same or different. As above, other nucleic acidanalogs may be used as well.

In addition, although the structures and discussion above depictsmetallocenes, and particularly ferrocene, this same general idea can beused to add ETMs in addition to metallocenes, as nucleoside replacementsor in polymer embodiments, described below. Thus, for example, when theETM is a transition metal complex other than a metallocene, comprisingone, two or three (or more) ligands, the ligands can be functionalizedas depicted for the ferrocene to allow the addition of phosphoramiditegroups. Particularly preferred in this embodiment are complexescomprising at least two ring (for example, aryl and substituted aryl)ligands, where each of the ligands comprises functional groups forattachment via phosphoramidite chemistry. As will be appreciated bythose in the art, this type of reaction; creating polymers of ETMseither as a portion of the backbone of the nucleic acid or as “sidegroups” of the nucleic acids, to allow amplification of the signalsgenerated herein, can be done with virtually any ETM that can befunctionalized to contain the correct chemical groups.

In addition, as is more fully outlined below, it is possible toincorporate more than one metallocene into the backbone, either withnucleotides in between and/or with adjacent metallocenes. When adjacentmetallocenes are added to the backbone, this is similar to the processdescribed below as “metallocene polymers”; that is, there are areas ofmetallocene polymers within the backbone.

In addition to the nucleic acid substitutent groups, it is alsodesirable in some instances to add additional substituent groups to oneor both of the aromatic rings of the metallocene (or ETM). Substituentgroups on an ETM, particularly metallocenes such as ferrocene, may beadded to alter the redox properties of the ETM. Thus, for example, insome embodiments, it may be desirable to have different ETMs attached indifferent ways (i.e. base or ribose attachment), on differentcomponents, or for different purposes (for example, calibration or as aninternal standard). Thus, the addition of substituent groups on themetallocene may allow two different ETMs to be distinguished.

In order to generate these metallocene-backbone nucleic acid analogs,the intermediate components are also provided. Thus, in a preferredembodiment, the invention provides phosphoramidite metallocenes, asgenerally depicted in Structure 28:

In Structure 28, PG is a protecting group, generally suitable for use innucleic acid synthesis, with DMT, MMT and TMT all being preferred. Thearomatic rings can either be the rings of the metallocene, or aromaticrings of ligands for transition metal complexes or other organic ETMs.The aromatic rings may be the same or different, and may be substitutedas discussed herein. Structure 29 depicts the ferrocene derivative:

These phosphoramidite analogs can be added to standard oligonucleotidesyntheses as is known in the art.

Structure 30 depicts the ferrocene peptide nucleic acid (PNA) monomer,that can be added to PNA synthesis as is known in the art and depictedwithin the Figures and Examples:

In Structure 30, the PG protecting group is suitable for use in peptidenucleic acid synthesis, with MMT, boc and Fmoc being preferred.

These same intermediate compounds can be used to form ETM or metallocenepolymers, which are added to the nucleic acids, rather than as backbonereplacements, as is more fully described below.

In a preferred embodiment, the ETMs are attached as polymers, forexample as metallocene polymers, in a “branched” configuration similarto the “branched DNA” embodiments herein and as outlined in U.S. Pat.No. 5,124,246, using modified functionalized nucleotides. The generalidea is as follows. A modified phosphoramidite nucleotide is generatedthat can ultimately contain a free hydroxy group that can be used in theattachment of phosphoramidite ETMs such as metallocenes. This freehydroxy group could be on the base or the backbone, such as the riboseor the phosphate (although as will be appreciated by those in the art,nucleic acid analogs containing other structures can also be used). Themodified nucleotide is incorporated into a nucleic acid, and any hydroxyprotecting groups are removed, thus leaving the free hydroxyl. Upon theaddition of a phosphoramidite ETM such as a metallocene, as describedabove in the Structures, ETMs, such as metallocene ETMs, are added.Additional phosphoramidite ETMs such as metallocenes can be added, toform “ETM polymers”, including “metallocene polymers” as depictedherein, particularly for ferrocene. In addition, in some embodiments, itis desirable to increase the solubility of the polymers by adding a“capping” group to the terminal ETM in the polymer, for example a finalphosphate group to the metallocene as is generally depicted in FIG. 4.Other suitable solubility enhancing “capping” groups will be appreciatedby those in the art. It should be noted that these solubility enhancinggroups can be added to the polymers in other places, including to theligand rings, for example on the metallocenes as discussed herein.

A preferred embodiment of this general idea is outlined in the Figures.In this embodiment, the 2′ position of a ribose of a phosphoramiditenucleotide is first functionalized to contain a protected hydroxy group,in this case via an oxo-linkage, although any number of linkers can beused, as is generally described herein for Z linkers. The protectedmodified nucleotide is then incorporated via standard phosphoramiditechemistry into a growing nucleic acid. The protecting group is removed,and the free hydroxy group is used, again using standard phosphoramiditechemistry to add a phosphoramidite metallocene such as ferrocene. Asimilar reaction is possible for nucleic acid analogs. For example,using peptide nucleic acids and the metallocene monomer shown inStructure 30, peptide nucleic acid structures containing metallocenepolymers could be generated.

Thus, the present invention provides recruitment linkers of nucleicacids comprising “branches” of metallocene polymers as is generallydepicted in FIGS. 3, 4 and 5. Preferred embodiments also utilizemetallocene polymers from one to about 50 metallocenes in length, withfrom about 5 to about 20 being preferred and from about 5 to about 10being especially preferred.

In addition, when the recruitment linker is nucleic acid, anycombination of ETM attachments may be done.

In a preferred embodiment, the recruitment linker is not nucleic acid,and instead may be any sort of linker or polymer. As will be appreciatedby those in the art, generally any linker or polymer that can bemodified to contain ETMs can be used. In general, the polymers orlinkers should be reasonably soluble and contain suitable functionalgroups for the addition of ETMs.

As used herein, a “recruitment polymer” comprises at least two or threesubunits, which are covalently attached. At least some portion of themonomeric subunits contain functional groups for the covalent attachmentof ETMs. In some embodiments coupling moieties are used to covalentlylink the subunits with the ETMs. Preferred functional groups forattachment are amino groups, carboxy groups, oxo groups and thiolgroups, with amino groups being particularly preferred. As will beappreciated by those in the art, a wide variety of recruitment polymersare possible.

Suitable linkers include, but are not limited to, alkyl linkers(including heteroalkyl (including (poly)ethylene glycol-typestructures), substituted alkyl, aryalkyl linkers, etc. As above for thepolymers, the linkers will comprise one or more functional groups forthe attachment of ETMs, which will be done as will be appreciated bythose in the art, for example through the use homo- orhetero-bifunctional linkers as are well known (see 1994 Pierce ChemicalCompany catalog, technical section on cross-linkers, pages 155-200,incorporated herein by reference).

Suitable recruitment polymers include, but are not limited to,functionalized styrenes, such as amino styrene, functionalized dextrans,and polyamino acids. Preferred polymers are polyamino acids (bothpoly-D-amino acids and poly-L-amino acids), such as polylysine, andpolymers containing lysine and other amino acids being particularlypreferred. Other suitable polyamino acids are polyglutamic acid,polyaspartic acid, co-polymers of lysine and glutamic or aspartic acid,co-polymers of lysine with alanine, tyrosine, phenylalanine, serine,tryptophan, and/or proline.

In a preferred embodiment, the recruitment linker comprises ametallocene polymer, as is described above.

When non-nucleic acid recruitment linkers are used, attachment of thelinker/polymer of the recruitment linker will be done generally usingstandard chemical techniques, such as will be appreciated by those inthe art. For example, when alkyl-based linkers are used, attachment canbe similar to the attachment of insulators to nucleic acids.

In addition, it is possible to have recruitment linkers that aremixtures of nucleic acids and non-nucleic acids, either in a linear form(i.e. nucleic acid segments linked together with alkyl linkers) or inbranched forms (nucleic acids with alkyl “branches” that may containETMs and may be additionally branched).

In a preferred embodiment, it is the target sequence itself that carriesthe ETMs. For example, as is more fully described below, it is possibleto enzymatically add triphosphate nucleotides comprising the ETMs of theinvention to a growing nucleic acid, for example during a polymerasechain reaction (PCR). As will be recognized by those in the art, whileseveral enzymes have been shown to generally tolerate modifiednucleotides, some of the modified nucleotides of the invention, forexample the “nucleoside replacement” embodiments and putatively some ofthe phosphate attachments, may or may not be recognized by the enzymesto allow incorporation into a growing nucleic acid. Therefore, preferredattachments in this embodiment are to the base or ribose of thenucleotide.

Thus, for example, PCR amplification of a target sequence, as is wellknown in the art, will result in target sequences comprising ETMs,generally randomly incorporated into the sequence. The system of theinvention can then be configured to allow detection using these ETMs.

Alternatively, as outlined more fully below, it is possible toenzymatically add nucleotides comprising ETMs to the terminus of anucleic acid, for example a target nucleic acid. In this embodiment, aneffective “recruitment linker” is added to the terminus of the targetsequence, that can then be used for detection.

In some embodiments, when the recruitment linker is nucleic acid, it maybe desirable in some instances to have some or all of the recruitmentlinker be double stranded. In one embodiment, there may be a secondrecruitment linker, substantially complementary to the first recruitmentlinker, that can hybridize to the first recruitment linker. In apreferred embodiment, the first recruitment linker comprises thecovalently attached ETMs. In an alternative embodiment, the secondrecruitment linker contains the ETMs, and the first recruitment linkerdoes not, and the ETMs are recruited to the surface by hybridization ofthe second recruitment linker to the first. In yet another embodiment,both the first and second recruitment linkers comprise ETMs. It shouldbe noted, as discussed above, that nucleic acids comprising a largenumber of ETMs may not hybridize as well, i.e. the T_(m) may bedecreased, depending on the site of attachment and the characteristicsof the ETM. Thus, in general, when multiple ETMs are used on hybridizingstrands, generally there are less than about 5, with less than about 3being preferred, or alternatively the ETMs should be spaced sufficientlyfar apart that the intervening nucleotides can sufficiently hybridize toallow good kinetics.

In a preferred embodiment, the reporter particle does not comprisedirectly attached ETMs. Rather, the reporter particle comprises twodifferent components: a capture binding partner (used to bind to theassay complex) and at least one amplification sequences, to which labelprobes will bind. This embodiment is generally depicted in FIG. 2H.

In this embodiment, the reporter particle comprises a capture bindingpartner as outlined above. In addition; the reporter particle comprisesnucleic acid probes comprising amplification sequences, to which labelprobes can bind. Again, for each of these, binding may be direct orindirect. An “amplification sequence” or “amplification segment” orgrammatical equivalents herein is meant a sequence that is used, eitherdirectly or indirectly, to bind to a first portion of a label probe; thelabel probe comprises the ETMs. Preferably, the reporter particlecomprises a multiplicity of amplification sequences.

The amplification sequences of the amplifier probe are used, eitherdirectly or indirectly, to bind to a label probe to allow detection. Ina preferred embodiment, the amplification sequences of the amplifierprobe are substantially complementary to a first portion of a labelprobe. Alternatively, amplifier extender probes are used, that have afirst portion that binds to the amplification sequence and a secondportion that binds to the first portion of the label probe.

Thus, label probes are either substantially complementary to anamplification sequence or to a portion of the target sequence.Accordingly, the label probes can be configured in a variety of ways, asis generally described herein, depending on whether a “mechanism-1” or“mechanism-2” detection system is utilized, as described below.

In a preferred embodiment, the reporter particle further comprises aSAM, as is outlined above for the transport particle and/or electrode.In general, this is a mixed monolayer comprising one, two, three or fourcomponents, as outlined above for electrodes and/or transport particles.

The transport moiety and the reporter composition may be added togetherin a wide variety of ratios, depending on the composition of themoieties (for example, whether they are both particles), the relativesize of the moieties, the density of the binding partners on theparticles, etc. For example, when a magnetic first particle is used anda colloid particle as the reporter particle, the reporter particles aregenerally added in excess. What is important is that neither thetransport composition nor the reporter composition is added in suchexcess that a single type of particle or composition will carry all thetarget analytes; the ratios are important to allow both the transportand reporter compositions to bind. However, in general, depending on thesize of the particles, generally an excess of reporter particles areadded, particularly when large magnetic particles are used with smallerreporter particles.

Accordingly, the methods of the invention provide for the detection oftarget analytes in a test sample. In general, the sample is added to thecompositions of the invention comprising transport and reportercompositions. The sample is added to the compositions under conditionswhereby the target analyte, if present, can bind to the first and secondbinding ligands to form an assay complex. “Assay complex” herein ismeant the collection of moieties, including the target analyte and thebinding ligands that contains at least one ETM and thus allowsdetection. The composition of the assay complex depends on the use ofthe different components outlined herein. The assay complex is thentransported to the electrode surface, comprising the conductiveoligomers, and the presence or absence of the ETMs is detected.

Transport of the assay complex using the transport composition may bedone in a wide variety of ways, as will be appreciated by those in theart, and will depend in part on the components of the system. Thus, forexample, when magnetic particles are used as the transport particles,the application of a magnetic field to bring the assay complexes andextra magnetic transport particles to the surface can be done. When thetransport particles are not magnetic, transport can proceed via physicalaggregation of the assay complexes with gravitational settling of theassay complexes on the electrode. Alternate transport mechanismsinclude, but are not limited to, electrophoretic transport of the assaycomplexes, centrifugation, etc. It should be noted that the use ofdifferent sized colloids may allow the use of differentialelectrophoretic transport based on different charge densities; that is,when a larger transport particle is used with smaller (charged) reporterparticles, i.e. the transport particle can be “covered” or “coated” withthe smaller particles, and thus the charge densities of the transportparticle and the assay complex will allow differential electrophoretictransport.

Probes of the present invention are designed to be complementary to atarget sequence (either the target sequence of the sample or to otherprobe sequences, as is described below), such that hybridization of thetarget sequence and the probes of the present invention occurs. Asoutlined below, this complementarity need not be perfect; there may beany number of base pair mismatches which will interfere withhybridization between the target sequence and the single strandednucleic acids of the present invention. However, if the number ofmutations is so great that no hybridization can occur under even theleast stringent of hybridization conditions, the sequence is not acomplementary target sequence. Thus, by “substantially complementary”herein is meant that the probes are sufficiently complementary to thetarget sequences to hybridize under normal reaction conditions.

A variety of hybridization conditions may be used in the presentinvention, including high, moderate and low stringency conditions; seefor example Maniatis et al., Molecular Cloning: A Laboratory Manual, 2dEdition, 1989, and Short Protocols in Molecular Biology, ed. Ausubel, etal, hereby incorporated by reference. Stringent conditions aresequence-dependent and will be different in different circumstances.Longer sequences hybridize specifically at higher temperatures. Anextensive guide to the hybridization of nucleic acids is found inTijssen, Techniques in Biochemistry and Molecular Biology—Hybridizationwith Nucleic Acid Probes, “Overview of principles of hybridization andthe strategy of nucleic acid assays” (1993). Generally, stringentconditions are selected to be about 5-10° C. lower than the thermalmelting point (Tm) for the specific sequence at a defined ionic strengthpH. The Tm is the temperature (under defined ionic strength, pH andnucleic acid concentration) at which 50% of the probes complementary tothe target hybridize to the target sequence at equilibrium (as thetarget sequences are present in excess, at Tm, 50% of the probes areoccupied at equilibrium). Stringent conditions will be those in whichthe salt concentration is less than about 1.0 sodium ion, typicallyabout 0.01 to 1.0 M sodium ion concentration (or other salts) at pH 7.0to 8.3 and the temperature is at least about 30° C. for short probes(e.g. 10 to 50 nucleotides) and at least about 60° C. for long probes(e.g. greater than 50 nucleotides). Stringent conditions may also beachieved with the addition of destabilizing agents such as formamide.The hybridization conditions may also vary when a non-ionic backbone,i.e. PNA is used, as is known in the art. In addition, cross-linkingagents may be added after target binding to cross-link, i.e. covalentlyattach, the two strands of the hybridization complex.

Thus, the assays are generally run under stringency conditions whichallows formation of the label probe hybridization complex only in thepresence of target. Stringency can be controlled by altering a stepparameter that is a thermodynamic variable, including, but not limitedto, temperature, formamide concentration, salt concentration, chaotropicsalt concentration pH, organic solvent concentration, etc.

These parameters may also be used to control non-specific binding, as isgenerally outlined in U.S. Pat. No. 5,681,697. Thus it may be desirableto perform certain steps at higher stringency conditions to reducenon-specific binding.

The reactions outlined herein may be accomplished in a variety of ways,as will be appreciated by those in the art. Components of the reactionmay be added simultaneously, or sequentially, in any order, withpreferred embodiments outlined below. In addition, the reaction mayinclude a variety of other reagents may be included in the assays. Theseinclude reagents like salts, buffers, neutral proteins, e.g. albumin,detergents, etc which may be used to facilitate optimal hybridizationand detection, and/or reduce non-specific or background interactions.Also reagents that otherwise improve the efficiency of the assay, suchas protease inhibitors, nuclease inhibitors, anti-microbial agents,etc., may be used, depending on the sample preparation methods andpurity of the target.

Once the assay complexes of the invention are made, that minimallycomprise a target analyte and at least one ETM, detection proceeds withelectronic initiation. Without being limited by the mechanism or theory,detection is based on the transfer of electrons from the ETM to theelectrode.

Detection of electron transfer, i.e. the presence of the ETMs, isgenerally initiated electronically, with voltage being preferred. Apotential is applied to the assay complex. Precise control andvariations in the applied potential can be via a potentiostat and eithera three electrode system (one reference, one sample (or working) and onecounter electrode) or a two electrode system (one sample and one counterelectrode). This allows matching of applied potential to peak potentialof the system which depends in part on the choice of ETMs and in part onthe conductive oligomer used, the composition and integrity of themonolayer, and what type of reference electrode is used. As describedherein, ferrocene is a preferred ETM.

Once the assay complexes of the invention are made, that minimallycomprise a target sequence and a label probe, detection proceeds withelectronic initiation. Without being limited by the mechanism or theory,detection is based on the transfer of electrons from the ETM to theelectrode.

Detection of electron transfer, i.e. the presence of the ETMs, isgenerally initiated electronically, with voltage being preferred. Apotential is applied to the assay complex. Precise control andvariations in the applied potential can be via a potentiostat and eithera three electrode system (one reference, one sample (or working) and onecounter electrode) or a two electrode system (one sample and one counterelectrode). This allows matching of applied potential to peak potentialof the system which depends in part on the choice of ETMs and in part onthe conductive oligomer used, the composition and integrity of themonolayer, and what type of reference electrode is used. As describedherein; ferrocene is a preferred ETM.

In a preferred embodiment, a co-reductant or co-oxidant (collectively,co-redoxant) is used, as an additional electron source or sink. Seegenerally Sato et al., Bull. Chem. Soc. Jpn 66:1032 (1993); Uosaki etal., Electrochimica Acta 36:1799 (1991); and Alleman et al., J. Phys.Chem. 100:17050 (1996); all of which are incorporated by reference.

In a preferred embodiment, an input electron source in solution is usedin the initiation of electron transfer, preferably when initiation anddetection are being done using DC current or at AC frequencies wherediffusion is not limiting. In general, as will be appreciated by thosein the art, preferred embodiments utilize monolayers that contain aminimum of “holes”, such that short-circuiting of the system is avoided.This may be done in several general ways. In a preferred embodiment, aninput electron source is used that has a lower or similar redoxpotential than the ETM of the label probe. Thus, at voltages above theredox potential of the input electron source, both the ETM and the inputelectron source are oxidized and can thus donate electrons; the ETMdonates an electron to the electrode and the input source donates to theETM. For example, ferrocene, as a ETM attached to the compositions ofthe invention as described in the examples, has a redox potential ofroughly 200 mV in aqueous solution (which can change significantlydepending on what the ferrocene is bound to, the manner of the linkageand the presence of any substitution groups). Ferrocyanide, an electronsource, has a redox potential of roughly 200 mV as well (in aqueoussolution). Accordingly, at or above voltages of roughly 200 mV,ferrocene is converted to ferricenium, which then transfers an electronto the electrode. Now the ferricyanide can be oxidized to transfer anelectron to the ETM. In this way, the electron source (or co-reductant)serves to amplify the signal generated in the system, as the electronsource molecules rapidly and repeatedly donate electrons to the ETMattached to the nucleic acid. The rate of electron donation oracceptance will be limited by the rate of diffusion of the co-reductant,the electron transfer between the co-reductant and the ETM, which inturn is affected by the concentration and size, etc.

Alternatively, input electron sources that have lower redox potentialsthan the ETM are used. At voltages less than the redox potential of theETM, but higher than the redox potential of the electron source, theinput source such as ferrocyanide is unable to be oxided and thus isunable to donate an electron to the ETM; i.e. no electron transferoccurs. Once ferrocene is oxidized, then there is a pathway for electrontransfer.

In an alternate preferred embodiment, an input electron source is usedthat has a higher redox potential than the ETM of the label probe. Forexample, luminol, an electron source, has a redox potential of roughly720 mV. At voltages higher than the redox potential of the ETM, butlower than the redox potential of the electron source, i.e. 200-720 mV,the ferrocene is oxided, and transfers a single electron to theelectrode via the conductive oligomer. However, the ETM is unable toaccept any electrons from the luminol electron source, since thevoltages are less than the redox potential of the luminol. However, ator above the redox potential of luminol, the luminol then transfers anelectron to the ETM, allowing rapid and repeated electron transfer. Inthis way, the electron source (or co-reductant) serves to amplify thesignal generated in the system, as the electron source molecules rapidlyand repeatedly donate electrons to the ETM of the label probe.

Luminol has the added benefit of becoming a chemiluminiscent speciesupon oxidation (see Jirka et al., Analytica Chimica Acta 284:345(1993)), thus allowing photo-detection of electron transfer from the ETMto the electrode. Thus, as long as the luminol is unable to contact theelectrode directly, i.e. in the presence of the SAM such that there isno efficient electron transfer pathway to the electrode, luminol canonly be oxidized by transferring an electron to the ETM on the labelprobe. When the ETM is not present, i.e. when the target sequence is nothybridized to the composition of the invention, luminol is notsignificantly oxidized, resulting in a low photon emission and thus alow (if any) signal from the luminol. In the presence of the target, amuch larger signal is generated. Thus, the measure of luminol oxidationby photon emission is an indirect measurement of the ability of the ETMto donate electrons to the electrode. Furthermore, since photondetection is generally more sensitive than electronic detection, thesensitivity of the system may be increased. Initial results suggest thatluminescence may depend on hydrogen peroxide concentration, pH, andluminol concentration, the latter of which appears to be non-linear.

Suitable electron source molecules are well known in the art, andinclude, but are not limited to, ferricyanide, and luminol.

Alternatively, output electron acceptors or sinks could be used, i.e.the above reactions could be run in reverse, with the ETM such as ametallocene receiving an electron from the electrode, converting it tothe metallicenium; with the output electron acceptor then accepting theelectron rapidly and repeatedly. In this embodiment, cobalticenium isthe preferred ETM.

The presence of the ETMs at the surface of the monolayer can be detectedin a variety of ways. A variety of detection methods may be used,including, but not limited to, optical detection (as a result ofspectral changes upon changes in redox states), which includesfluorescence, phosphorescence, luminiscence, chemiluminescence,electrochemiluminescence, and refractive index; and electronicdetection, including, but not limited to, amperommetry, voltammetry,capacitance and impedence. These methods include time or frequencydependent methods based on AC or DC currents, pulsed methods, lock-intechniques, filtering (high pass, low pass, band pass), andtime-resolved techniques including time-resolved fluoroscence.

In one embodiment, the efficient transfer of electrons from the ETM tothe electrode results in stereotyped changes in the redox state of theETM. With many ETMs including the complexes of ruthenium containingbipyridine, pyridine and imidazole rings, these changes in redox stateare associated with changes in spectral properties. Significantdifferences in absorbance are observed between reduced and oxidizedstates for these molecules. See for example Fabbrizzi et al., Chem. Soc.Rev. 1995 pp 197-202). These differences can be monitored using aspectrophotometer or simple photomultiplier tube device.

In this embodiment, possible electron donors and acceptors include allthe derivatives listed above for photoactivation or initiation.Preferred electron donors and acceptors have characteristically largespectral changes upon oxidation and reduction resulting in highlysensitive monitoring of electron transfer. Such examples includeRu(NH₃)₄py and Ru(bpy)₂im as preferred examples. It should be understoodthat only the donor or acceptor that is being monitored by absorbanceneed have ideal spectral characteristics.

In a preferred embodiment, the electron transfer is detectedfluorometrically. Numerous transition metal complexes, including thoseof ruthenium, have distinct fluorescence properties. Therefore, thechange in redox state of the electron donors and electron acceptorsattached to the nucleic acid can be monitored very sensitively usingfluorescence, for example with Ru(4,7-biphenyl₂-phenanthroline)₃ ²⁺. Theproduction of this compound can be easily measured using standardfluorescence assay techniques. For example, laser induced fluorescencecan be recorded in a standard single cell fluorimeter, a flow through“on-line” fluorimeter (such as those attached to a chromatographysystem) or a multi-sample “plate-reader” similar to those marketed for96-well immuno assays.

Alternatively, fluorescence can be measured using fiber optic sensorswith nucleic acid probes in solution or attached to the fiber optic.Fluorescence is monitored using a photomultiplier tube or other lightdetection instrument attached to the fiber optic. The advantage of thissystem is the extremely small volumes of sample that can be assayed.

In addition, scanning fluorescence detectors such as the FluorImagersold by Molecular Dynamics are ideally suited to monitoring thefluorescence of modified nucleic acid molecules arrayed on solidsurfaces. The advantage of this system is the large number of electrontransfer probes that can be scanned at once using chips covered withthousands of distinct nucleic acid probes.

Many transition metal complexes display fluorescence with large Stokesshifts. Suitable examples include bis- and trisphenanthroline complexesand bis- and trisbipyridyl complexes of transition metals such asruthenium (see Juris, A., Balzani, V., et. al. Coord. Chem. Rev., V. 84,p. 85-277, 1988). Preferred examples display efficient fluorescence(reasonably high quantum yields) as well as low reorganization energies.These include Ru(4,7-biphenyl₂-phenanthroline)₃ ²⁺,Ru(4,4′-diphenyl-2,2′-bipyridine)₃ ²⁺ and platinum complexes (seeCummings et al., J. Am. Chem. Sob. 118:1949-1960 (1996), incorporated byreference). Alternatively, a reduction in fluorescence associated withhybridization can be measured using these systems.

In a further embodiment, electrochemiluminescence is used as the basisof the electron transfer detection. With some ETMs such as Ru²⁺(bpy)₃,direct luminescence accompanies excited state decay. Changes in thispropertyare associated with nucleic acid hybridization and can bemonitored with a simple photomultiplier tube arrangement (see.Blackburn, G. F. Clin. Chem. 37: 1534-1539 (1991); and Juris et al.,supra.

In a preferred embodiment, electronic detection is used, includingamperommetry, voltammetry, capacitance, and impedence. Suitabletechniques include, but are not limited to, electrogravimetry;coulometry (including controlled potential coulometry and constantcurrent coulometry); voltametry (cyclic voltametry, pulse voltametry(normal pulse voltametry, square wave voltametry, differential pulsevoltametry, Osteryoung square wave voltametry, and coulostatic pulsetechniques); stripping analysis (aniodic stripping analysis, cathiodicstripping analysis, square wave stripping voltammetry); conductancemeasurements (electrolytic conductance, direct analysis); time-dependentelectrochemical analyses (chronoamperometry, chronopotentiometry, cyclicchronopotentiometry and amperometry, AC polography, chronogalvametry,and chronocoulometry); AC impedance measurement; capacitancemeasurement; AC voltametry; and photoelectrochemistry.

In a preferred embodiment, monitoring electron transfer is viaamperometric detection. This method of detection involves applying apotential (as compared to a separate reference electrode) between thenucleic acid-conjugated electrode and a reference (counter) electrode inthe sample containing target genes of interest. Electron transfer ofdiffering efficiencies is induced in samples in the presence or absenceof target nucleic acid; that is, the presence or absence of the targetnucleic acid, and thus the label probe, can result in differentcurrents.

The device for measuring electron transfer amperometrically involvessensitive current detection and includes a means of controlling thevoltage potential, usually a potentiostat. This voltage is optimizedwith reference to the potential of the electron donating complex on thelabel probe. Possible electron donating complexes include thosepreviously mentioned with complexes of iron, osmium, platinum, cobalt,rhenium and ruthenium being preferred and complexes of iron being mostpreferred.

In a preferred embodiment, alternative electron detection modes areutilized. For example, potentiometric (or voltammetric) measurementsinvolve non-faradaic (no net current flow) processes and are utilizedtraditionally in pH and other ion detectors. Similar sensors are used tomonitor electron transfer between the ETM and the electrode. Inaddition, other properties of insulators (such as resistance) and ofconductors (such as conductivity, impedance and capicitance) could beused to monitor electron transfer between ETM and the electrode.Finally, any system that generates a current (such as electron transfer)also generates a small magnetic field, which may be monitored in someembodiments.

It should be understood that one benefit of the fast rates of electrontransfer observed in the compositions of the invention is that timeresolution can greatly enhance the signal-to-noise results of monitorsbased on absorbance, fluorescence and electronic current. The fast ratesof electron transfer of the present invention result both in highsignals and stereotyped delays between electron transfer initiation andcompletion. By amplifying signals of particular delays, such as throughthe use of pulsed initiation of electron transfer and “lock-in”amplifiers of detection, and Fourier transforms.

In a preferred embodiment, electron transfer is initiated usingalternating current (AC) methods. Without being bound by theory, itappears that ETMs, bound to an electrode, generally respond similarly toan AC voltage across a circuit containing resistors and capacitors.Basically, any methods which enable the determination of the nature ofthese complexes, which act as a resistor and capacitor, can be used asthe basis of detection. Surprisingly, traditional electrochemicaltheory, such as exemplified in Laviron et al., J. Electroanal. Chem.97:135 (1979) and Laviron et al., J. Electroanal. Chem. 105:35 (1979),both of which are incorporated by reference, do not accurately model thesystems described herein, except for very small E_(AC) (less than 10 mV)and relatively large numbers of molecules. That is, the AC current (1)is not accurately described by Laviron's equation. This may be due inpart to the fact that this theory assumes an unlimited source and sinkof electrons, which is not true in the present systems.

The AC voltametry theory that models these systems well is outlined inO'Connor et al., J. Electroanal. Chem. 466(2):197-202 (1999), herebyexpressly incorporated by reference. The equation that predicts thesesystems is shown below as Equation 1:

$\begin{matrix}{i_{avg} = {2\; {{nfFN}_{total} \cdot \frac{\sinh \left\lbrack {\frac{nF}{RT} \cdot E_{AC}} \right\rbrack}{{\cosh \left\lbrack {\frac{nF}{RT} \cdot E_{AC}} \right\rbrack} + {\cosh \left\lbrack {\frac{nF}{RT}\left( {E_{DC} - E_{O}} \right)} \right\rbrack}}}}} & {{Equation}\mspace{14mu} 1}\end{matrix}$

In Equation 1, n is the number of electrons oxidized or reduced perredox molecule, f is the applied frequency, F is Faraday's constant,N_(total) is the total number of redox molecules, E_(O) is the formalpotential of the redox molecule, R is the gas constant, T is thetemperature in degrees Kelvin, and E_(DC) is the electrode potential.The model fits the experimental data very well. In some cases thecurrent is smaller than predicted, however this has been shown to becaused by ferrocene degradation which may be remedied in a number ofways.

In addition, the faradaic current can also be expressed as a function oftime, as shown in Equation 2:

$\begin{matrix}{{I_{f}(t)} = {\frac{q_{e}N_{total}{nF}}{2\; {{RT}\left( {{\cosh \left\lbrack {\frac{nF}{RT}\left( {{V(t)} - E_{0}} \right)} \right\rbrack} + 1} \right)}} \cdot \frac{{V(t)}}{t}}} & {{Equation}\mspace{14mu} 2}\end{matrix}$

I_(F) is the Faradaic current and q_(e) is the elementary charge.

However, Equation 1 does not incorporate the effect of electron transferrate nor of instrument factors. Electron transfer rate is important whenthe rate is close to or lower than the applied frequency. Thus, the truei_(AC) should be a function of all three, as depicted in Equation 3.

i _(AC) =f(Nernst factors)f(k _(ET))f(instrument factors)  Equation 3

These equations can be used to model and predict the expected ACcurrents in systems which use input signals comprising both AC and DCcomponents. As outlined above, traditional theory surprisingly does notmodel these systems at all, except for very low voltages.

In general, non-specifically bound label probes/ETMs show differences inimpedance (i.e. higher impedances) than when the label probes containingthe ETMs are specifically bound in the correct orientation. In apreferred embodiment, the non-specifically bound material is washedaway, resulting in an effective impedance of infinity. Thus, ACdetection gives several advantages as is generally discussed below,including an increase in sensitivity, and the ability to “filter out”background noise. In particular, changes in impedance (including, forexample, bulk impedance) as between non-specific binding ofETM-containing probes and target-specific assay complex formation may bemonitored.

Accordingly, when using AC initiation and detection methods, thefrequency response of the system changes as a result of the presence ofthe ETM. By “frequency response” herein is meant a modification ofsignals as a result of electron transfer between the electrode and theETM. This modification is different depending on signal frequency. Afrequency response includes AC currents at one or more frequencies,phase shifts, DC offset voltages, faradaic impedance, etc.

Once the assay complex including the target sequence and label probe ismade, a first input electrical signal is then applied to the system,preferably via at least the sample electrode (containing the complexesof the invention) and the counter electrode, to initiate electrontransfer between the electrode and the ETM. Three electrode systems mayalso be used, with the voltage applied to the reference and workingelectrodes. The first input signal comprises at least an AC component.The AC component may be of variable amplitude and frequency. Generally,for use in the present methods, the AC amplitude ranges from about 1 mVto about 1.1 V, with from about 10 mV to about 800 mV being preferred,and from about 10 mV to about 500 mV being especially preferred. The ACfrequency ranges from about 0.01 Hz to about 100 MHz, with from about 10Hz to about 10 MHz being preferred, and from about 100 Hz to about 20MHz being especially preferred.

The use of combinations of AC and DC signals gives a variety ofadvantages, including surprising sensitivity and signal maximization.

In a preferred embodiment, the first input signal comprises a DCcomponent and an AC component. That is, a DC offset voltage between thesample and counter electrodes is swept through the electrochemicalpotential of the ETM (for example, when ferrocene is used, the sweep isgenerally from 0 to 500 mV) (or alternatively, the working electrode isgrounded and the reference electrode is swept from 0 to −500 mV). Thesweep is used to identify the DC voltage at which the maximum responseof the system is seen. This is generally at or about the electrochemicalpotential of the ETM. Once this voltage is determined, either a sweep orone or more uniform DC offset voltages may be used: DC offset voltagesof from about −1 V to about +1.1 V are preferred, with from about −500mV to about +800 mV being especially preferred, and from about −300 mVto about 500 mV being particularly preferred. In a preferred embodiment,the DC offset voltage is not zero. On top of the DC offset voltage, anAC signal component of variable amplitude and frequency is applied. Ifthe ETM is present, and can respond to the AC perturbation, an ACcurrent will be produced due to electron transfer between the electrodeand the ETM.

For defined systems, it may be sufficient to apply a single input signalto differentiate between the presence and absence of the ETM (i.e. thepresence of the target sequence) nucleic acid. Alternatively, aplurality of input signals are applied. As outlined herein, this maytake a variety of forms, including using multiple frequencies, multipleDC offset voltages, or multiple AC amplitudes, or combinations of any orall of these.

Thus, in a preferred embodiment, multiple DC offset voltages are used,although as outlined above, DC voltage sweeps are preferred. This may bedone at a single frequency, or at two or more frequencies.

In a preferred embodiment, the AC amplitude is varied. Without beingbound by theory, it appears that increasing the amplitude increases thedriving force. Thus, higher amplitudes, which result in higheroverpotentials give faster rates of electron transfer. Thus, generally,the same system gives an improved response (i.e. higher output signals)at any single frequency through the use of higher overpotentials at thatfrequency. Thus, the amplitude may be increased at high frequencies toincrease the rate of electron transfer through the system, resulting ingreater sensitivity. In addition, this may be used, for example, toinduce responses in slower systems such as those that do not possessoptimal spacing configurations.

In a preferred embodiment, measurements of the system are taken at leasttwo separate amplitudes or overpotentials, with measurements at aplurality of amplitudes being preferred. As noted above, changes inresponse as a result of changes in amplitude may form the basis ofidentification, calibration and quantification of the system. Inaddition, one or more AC frequencies can be used as well.

In a preferred embodiment, the AC frequency is varied. At differentfrequencies, different molecules respond in different ways. As will beappreciated by those in the art, increasing the frequency generallyincreases the output current. However, when the frequency is greaterthan the rate at which electrons may travel between the electrode andthe ETM, higher frequencies result in a loss or decrease of outputsignal. At some point, the frequency will be greater than the rate ofelectron transfer between the ETM and the electrode, and then the outputsignal will also drop.

In one embodiment, detection utilizes a single measurement of outputsignal at a single frequency. That is, the frequency response of thesystem in the absence of target sequence, and thus the absence of labelprobe containing ETMs, can be previously determined to be very low at aparticular high frequency. Using this information, any response at aparticular frequency, will show the presence of the assay complex. Thatis, any response at a particular frequency is characteristic of theassay) complex. Thus, it may only be necessary to use a single inputhigh frequency, and any changes in frequency response is an indicationthat the ETM is present, and thus that the target sequence is present.

In addition, the use of AC techniques allows the significant reductionof background signals at any single frequency due to entities other thanthe ETMs, i.e. “locking out” or “filtering” unwanted signals. That is,the frequency response of a charge carrier or redox active molecule insolution will be limited by its diffusion coefficient and chargetransfer coefficient. Accordingly, at high frequencies, a charge carriermay not diffuse rapidly enough to transfer its charge to the electrode,and/or the charge transfer kinetics may not be fast enough. This isparticularly significant in embodiments that do not have goodmonolayers, i.e. have partial or insufficient monolayers, i.e. where thesolvent is accessible to the electrode. As outlined above, in DCtechniques, the presence of “holes” where the electrode is accessible tothe solvent can result in solvent charge carriers “short circuiting” thesystem, i.e. the reach the electrode and generate background signal.However, using the present AC techniques, one or more frequencies can bechosen that prevent a frequency response of one or more charge carriersin solution, whether or not a monolayer is present. This is particularlysignificant since many biological fluids such as blood containsignificant amounts of redox active molecules which can interfere withamperometric detection methods.

In a preferred embodiment, measurements of the system are taken at leasttwo separate frequencies, with measurements at a plurality offrequencies being preferred. A plurality of frequencies includes a scan.For example, measuring the output signal, e.g., the AC current, at a lowinput frequency such as 1-20 Hz, and comparing the response to theoutput signal at high frequency such as 10-100 kHz will show a frequencyresponse difference between the presence and absence of the ETM. In apreferred embodiment, the frequency response is determined at least two,preferably at least about five, and more preferably at least about tenfrequencies.

After transmitting the input signal to initiate electron transfer, anoutput signal is received or detected. The presence and magnitude of theoutput signal will depend on a number of factors, including theoverpotential/amplitude of the input signal; the frequency of the inputAC signal; the composition of the intervening medium; the DC offset; theenvironment of the system; the nature of the ETM; the solvent; and thetype and concentration of salt. At a given input signal, the presenceand magnitude of the output signal will depend in general on thepresence or absence of the ETM, the placement and distance of the ETMfrom the surface of the monolayer and the character of the input signal.In some embodiments, it may be possible to distinguish betweennon-specific binding of label probes and the formation of targetspecific assay complexes containing label probes, on the basis ofimpedance.

In a preferred embodiment, the output signal comprises an AC current. Asoutlined above, the magnitude of the output current will depend on anumber of parameters. By varying these parameters, the system may beoptimized in a number of ways.

In general, AC currents generated in the present invention range fromabout 1 femptoamp to about 1 milliamp, with currents from about 50femptoamps to about 100 microamps being preferred, and from about 1picoamp to about 1 microamp being especially preferred.

In a preferred embodiment, the output signal is phase shifted in the ACcomponent relative to the input signal. Without being bound by theory,it appears that the systems of the present invention may be sufficientlyuniform to allow phase-shifting based detection. That is, the complexbiomolecules of the invention through which electron transfer occursreact to the AC input in a homogeneous manner, similar to standardelectronic components, such that a phase shift can be determined. Thismay serve as the basis of detection between the presence and absence ofthe ETM, and/or differences between the presence of target-specificassay complexes comprising label probes and non-specific binding of thelabel probes to the system components.

The output signal is characteristic of the presence of the ETM; that is,the output signal is characteristic of the presence of thetarget-specific assay complex comprising label probes and ETMs. In apreferred embodiment, the basis of the detection is a difference in thefaradaic impedance of the system as a result of the formation of theassay complex. Faradaic impedance is the impedance of the system betweenthe electrode and the ETM. Faradaic impedance is quite different fromthe bulk or dielectric impedance, which is the impedance of the bulksolution between the electrodes. Many factors may change the faradaicimpedance which may not effect the bulk impedance, and vice versa. Thus,the assay complexes comprising the nucleic acids in this system have acertain faradaic impedance, that will depend on the distance between theETM and the electrode, their electronic properties, and the compositionof the intervening medium, among other things. Of importance in themethods of the invention is that the faradaic impedance between the ETMand the electrode is signficantly different depending on whether thelabel probes containing the ETMs are specifically or non-specificallybound to the electrode.

Accordingly, the present invention further provides apparatus for thedetection of nucleic acids using AC detection methods. The apparatusincludes a test chamber which has at least a first measuring or sampleelectrode, and a second measuring or counter electrode. Three electrodesystems are also useful. The first and second measuring electrodes arein contact with a test sample receiving region, such that in thepresence of a liquid test sample, the two electrodes may be inelectrical contact.

In a preferred embodiment, the first measuring electrode comprises asingle stranded nucleic acid capture probe covalently attached via anattachment linker, and a monolayer comprising conductive oligomers, suchas are described herein.

The apparatus further comprises an AC voltage source electricallyconnected to the test chamber; that is, to the measuring electrodes.Preferably, the AC voltage source is capable of delivering DC offsetvoltage as well.

In a preferred embodiment, the apparatus further comprises a processorcapable of comparing the input signal and the output signal. Theprocessor is coupled to the electrodes and configured to receive anoutput signal, and thus detect the presence of the target nucleic acid.

Thus, the compositions of the present invention may be used in a varietyof research, clinical, quality control, or field testing settings.

In a preferred embodiment, the probes are used in genetic diagnosis. Forexample, probes can be made using the techniques disclosed herein todetect target sequences such as the gene for nonpolyposis colon cancer,the BRCA1 breast cancer gene, P53, which is a gene associated with avariety of cancers, the Apo E4 gene that indicates a greater risk ofAlzheimer's disease, allowing for easy presymptomatic screening ofpatients, mutations in the cystic fibrosis gene, or any of the otherswell known in the art.

In an additional embodiment, viral and bacterial detection is done usingthe complexes of the invention. In this embodiment, probes are designedto detect target sequences from a variety of bacteria and viruses. Forexample, current blood-screening techniques rely on the detection ofanti-HIV antibodies. The methods disclosed herein allow for directscreening of clinical samples to detect HIV nucleic acid sequences,particularly highly conserved HIV sequences. In addition, this allowsdirect monitoring of circulating virus within a patient as an improvedmethod of assessing the efficacy of anti-viral therapies. Similarly,viruses associated with leukemia, HTLV-I and HTLV-II, may be detected inthis way. Bacterial infections such as tuberculosis, clymidia and othersexually transmitted diseases, may also be detected, for example usingribosomal RNA (rRNA) as the target sequences.

In a preferred embodiment, the nucleic acids of the invention find useas probes for toxic bacteria in the screening of water and food samples.For example, samples may be treated to lyse the bacteria to release itsnucleic acid (particularly rRNA), and then probes designed to recognizebacterial strains, including, but not limited to, such pathogenicstrains as, Salmonella, Campylobacter, Vibrio cholerae, Leishmania,enterotoxic strains of E. coli, and Legionnaire's disease bacteria.Similarly, bioremediation strategies may be evaluated using thecompositions of the invention.

In a further embodiment, the probes are used for forensic “DNAfingerprinting” to match crime-scene DNA against samples taken fromvictims and suspects.

In an additional embodiment, the probes in an array are used forsequencing by hybridization.

Thus, the present invention provides for extremely specific andsensitive probes, which may, in some embodiments, detect targetsequences without removal of unhybridized probe. This will be useful inthe generation of automated gene probe assays.

Alternatively, the compositions of the invention are useful to detectsuccessful gene amplification in PCR, thus allowing successful PCRreactions to be an indication of the presence or absence of a targetsequence. PCR may be used in this manner in several ways. For example,in one embodiment, the PCR reaction is done as is known in the art, andthen added to a composition of the invention comprising the targetnucleic acid with a ETM, covalently attached to an electrode via aconductive oligomer with subsequent detection of the target sequence.Alternatively, PCR is done using nucleotides labelled with a ETM, eitherin the presence of, or with subsequent addition to, an electrode with aconductive oligomer and a target nucleic acid. Binding of the PCRproduct containing ETMs to the electrode composition will allowdetection via electron transfer. Finally, the nucleic acid attached tothe electrode via a conductive polymer may be one PCR primer, withaddition of a second primer labelled with an ETM. Elongation results indouble stranded nucleic acid with a ETM and electrode covalentlyattached. In this way, the present invention is used for PCR detectionof target sequences.

In a preferred embodiment, the arrays are used for mRNA detection. Apreferred embodiment utilizes either capture probes or capture extenderprobes that hybridize close to the 3′ polyadenylation tail of the mRNAs.This allows the use of one species of target binding probe fordetection, i.e. the probe contains a poly-T portion that will bind tothe poly-A tail of the mRNA target. Generally, the probe will contain asecond portion, preferably non-poly-T, that will bind to the detectionprobe (or other probe). This allows one target-binding probe to be made,and thus decreases the amount of different probe synthesis that is done.

In a preferred embodiment, the use of restriction enzymes and ligationmethods allows the creation of “universal” arrays. In this embodiment,monolayers comprising capture probes that comprise restrictionendonuclease ends, as is generally depicted in FIG. 7 of PCT US97/20014.By utilizing complementary portions of nucleic acid, while leaving“sticky ends”, an array comprising any number of restrictionendonuclease sites is made. Treating a target sample with one or more ofthese restriction endonucleases allows the targets to bind to the array.This can be done without knowing the sequence of the target. The targetsequences can be ligated, as desired, using standard methods such asligases, and the target sequence detected, using either standard labelsor the methods of the invention.

The present invention provides methods which can result in sensitivedetection of nucleic acids. In a preferred embodiment, less than about10×10⁶ molecules are detected, with less than about 10×10⁵ beingpreferred, less than 10×10⁴ being particularly preferred, less thanabout 10×10³ being especially preferred, and less than about 10×10²being most preferred. As will be appreciated by those in the art, thisassumes a 1:1 correlation between target sequences and reportermolecules; if more than one reporter molecule (i.e. electron transferMoeity) is used for each target sequence, the sensitivity will go up.

All references cited herein are incorporated by reference in theirentirety.

1-6. (canceled)
 7. A method of detecting the presence of a targetanalyte comprising: a. providing an electrode comprising: i. Acovalently attached a first capture binding ligand; ii. A colloidalparticle comprising a second capture binding ligand; and iii. A targetanalyte, bound to said first and said second ligands; b. detecting anelectronic signal from said electrode as an indication of the presenceof said target analyte.
 8. A method according to claim 7 wherein saidfirst and said second capture binding ligands are nucleic acid sequencesand said target analyte is a nucleic acid sequence.
 9. A methodaccording to claim 7 or 8 wherein said colloidal particle comprises amaterial selected from the group consisting of Au, Se, Te, Co, Ni, Fe,Cu and Pt.
 10. A method according to claim 9 wherein said colloidalparticle comprises Au.
 11. A method according to claim 7 wherein saiddetecting utilizes AC voltametry.
 12. A method of detecting the presenceof a target analyte comprising: a. providing an array of electrodes,each electrode comprising: i. a covalently attached first nucleic acidprobe; ii. a colloidal particle comprising a second nucleic acid probe;and iii. a target nucleic acid, hybridized to said first and said secondnucleic acid probes; b. detecting a signal from said electrode as anindication of the presence of said target analyte.
 13. A methodaccording to claim 7 wherein said signal is electrical.
 14. A methodaccording to claim 7 wherein said signal is optical.
 15. A methodaccording to claim 12 wherein said colloidal particle comprises Au. 16.A composition comprising colloids comprising self-assembled monolayers.17. A composition according to claim 16 wherein said colloids are Au.18. A composition according to claim 17 wherein said self assembledmonolayers comprise thiol groups.
 19. A composition according to claim16 wherein said colloids further comprise capture binding ligands.
 20. Acomposition comprising: a. An electrode comprising a first capturebinding ligand; b. A colloidal particle comprising a second capturebinding ligand; and c. A target analyte, bound to said first and saidsecond ligands.
 21. A composition according to claim 20 wherein saidfirst and second capture binding ligands are capture probes, and saidtarget analyte is a target nucleic acid.
 22. A composition according toclaim 20 wherein said composition comprises an array of electrodes eachcomprising said first and second capture binding ligands and a targetanalyte.
 23. A method of detecting a target analyte comprisingcontacting a sample comprising said target analyte with a compositioncomprising: a. An electrode comprising a first capture binding ligand;b. A colloidal particle comprising a second capture binding ligand; andc. A target analyte, bound to said first and said second ligands; andand determining the presence or absence of said target analyte.